Guanine analogs as telomerase substrates and telomere length affectors

ABSTRACT

This invention relates to compounds useful for inhibiting telomere elongation. More specifically, the invention provides nucleotide analogs that are incorporated into telomeres by telomerase thereby inhibiting elongation of telomeres. The compounds are use in treating cancer and other cell proliferative diseases.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/579,575, filed Dec. 22, 2011, which is incorporated herein byreference in its entirety.

FIELD OF INVENTION

This invention relates to compounds useful for inhibiting telomereelongation. More specifically, the invention provides nucleotide analogsthat are incorporated into telomeres by telomerase thereby inhibitingelongation of telomeres. The compounds are useful in treating cancer andother cell proliferative diseases.

BACKGROUND

Telomerase is a ribonucleoprotein that catalyzes the addition oftelomeric repeat sequences to chromosome ends causing telomereelongation. See Blackburn, 1992, Ann. Rev. Biochem., 61:113-129. Thereis an extensive body of literature describing the connection betweentelomeres, telomerase, cellular senescence and cancer (for a generalreview, see Oncogene, vol. 21, January 2002, which is an entire issue ofthe journal focused on telomerase).

Genes encoding both the protein and RNA components of human telomerasehave been cloned and sequenced (see U.S. Pat. Nos. 6,261,836 and5,583,016, respectively) and much effort has been spent in the searchfor telomerase inhibitors. Telomerase inhibitors identified to dateinclude small molecule compounds and oligonucleotides. Variouspublications describe the use of oligonucleotides to inhibit telomerase,either targeted against the mRNA encoding the telomerase proteincomponent (the human form of which is known as human telomerase reversetranscriptase or hTERT) or the RNA component of the telomeraseholoenzyme (the human form of which is known as human telomerase RNA orhTR). Oligonucleotides that are targeted to the hTERT mRNA are generallybelieved to act as conventional antisense drugs in that they bind to themRNA, resulting in destruction of the mRNA, and thereby preventingproduction of the hTERT protein (see, for example, U.S. Pat. No.6,444,650). Certain oligonucleotides that are targeted to hTR aredesigned to bind to hTR molecules present within the telomeraseholoenzyme, and thereby disrupt enzyme function (see, for example, U.S.Pat. No. 6,548,298).

Given the close connection between telomerase and cell proliferativedisorders such as cancer, what is needed, therefore, are compoundsuseful for inhibiting telomere elongation in proliferative cells anduses of the same to treat disease.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles) are referenced. Thedisclosure of all patents, patent applications, and publications citedherein are hereby incorporated by reference in their entirety for allpurposes.

SUMMARY

The present invention relates to, inter alia, phosphonomethoxy guanosineanalogs, including prodrugs, suitable for use in the efficient oraldelivery of such analogs as well as uses of phosphonomethoxy guanosineanalogs for the inhibition of telomere chain elongation and thetreatment of cell proliferative disorders in individuals in needthereof.

The compositions and methods described herein inhibit the extension oftelomeres by telomerase. The compounds and methods also inhibitproliferation of cancer cells.

In accordance with this invention, compounds are provided having formula(VIII)

wherein G is selected from guanine-9-yl, or its 1-deaza or 3-deazaanalogs, Y independently is —OH, —NH(CH₂)_(n)NH(CH₂)_(n)NHR³; or—N[(CH₂)_(n)NH₂](CH₂)_(n)NHR³; R³ is —H or —(CH₂)NH₂; n independently is2-4; with the proviso that at least one Y is—NH(CH₂)_(n)NH(CH₂)_(n)NHR³; or —N[(CH₂)_(n)NH₂](CH₂)_(n)NHR³; and thesalts, hydrates, tautomers and solvates thereof. In one embodiment G isguanine-9-yl.

In one embodiment, at least one Y is —NH(CH₂)_(n)NH(CH₂)_(n)NHR³, R³ is—H or —(CH₂) NH₂ and n independently is 2-4.

In one embodiment, at least one Y is —NH(CH₂)₃NH(CH₂)₄NH₂ or—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂. In one embodiment one Y is—NH(CH₂)₃NH(CH₂)₄NH₂ and the other Y is —OH. In one embodiment one Y is—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ and the other Y is —OH. In one embodiment,both Ys are —NH(CH₂)₃NH(CH₂)₄NH₂. In one embodiment both Ys are—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂.

In one embodiment, at least one Y is —N[(CH₂)NH₂](CH₂)NHR³, R³ is —H or—(CH₂)_(n)NH₂ and n independently is 2-4.

In one embodiment, the compound of formula VIII is the enriched orisolated (R) enantiomer. In another embodiment, the compound of formulaVIII is the enriched or isolated (S) enantiomer.

In accordance with this invention, pharmaceutical compositions areprovided comprising the compounds of Formula VIII with apharmaceutically acceptable excipient.

In accordance with this invention, methods are provided for inhibitingtelomere elongation comprising contacting a cell with the compounds ofFormula VIII of this invention or the pharmaceutical compositions ofthis invention. In some embodiments, the cell is a cancer cell.

In accordance with this invention, methods are provided for shorteningtelomere length in a cell or tissue comprising contacting the cell ortissue with the compounds of Formula VIII of this invention or thepharmaceutical compositions of this invention.

In accordance with this invention, methods are provided for treatingcancer in a patient by administering an effective amount of thecompounds of Formula VIII of this invention or the pharmaceuticalcompositions of this invention to the patient. In one embodiment, thecancer is metastatic cancer. In one embodiment, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma).

In accordance with this invention, methods are provided for treating apatient by administering an effective amount of the compounds of FormulaVIII of this invention or the pharmaceutical compositions of thisinvention wherein the method involves oral, intra-arterial, intranasal,intraperitoneal, intravenous, intramuscular, subcutaneous, ortransdermal administration of the compound or the pharmaceuticalcompositions of the invention.

In accordance with this invention, methods are provided using compoundshaving formula (IX):

wherein G is selected from guanine-9-yl, or its 1-deaza or 3-deazaanalogs, X independently is —OH, a monophosphate, a diphosphate or—OCH(R¹)OC(O)OR¹, R¹ independently is —H, or C₁-C₅ alkyl; and the salts,hydrates, tautomers and solvates thereof; under conditions whereintelomere elongation is inhibited. In one embodiment, the compounds ofFormula IX wherein at least one X is —OCH₂OC(O)OR¹ and R¹ is C₁-C₅alkyl. In one embodiment, the compounds of Formula IX wherein one X is—OH and the other X is —OCH₂OC(O)OR¹ and R¹ is C₁-C₅ alkyl. In oneembodiment, the compounds of Formula IX wherein one X is —OH and theother X is —OCH₂OC(O)OCH(CH₃)₂. In another embodiment both Xs are—OCH₂OC(O)OCH(CH₃)₂. In another embodiment, one X is —OH and the other Xis diphosphate. A specific compound of Formula IX is9-[2-(phosphonomethoxy)propyl]-guanine diisopropyloxy ester, or apharmaceutically acceptable salt thereof. A specific compound of FormulaVIII is (R)-9-[2-(phosphonomethoxy)propyl]-guanine diisopropyloxy ester,or a pharmaceutically acceptable salt thereof. A specific compound ofFormula IX is (S)-9-[2-(phosphonomethoxy)propyl]-guanine diisopropyloxyester, or a pharmaceutically acceptable salt thereof. A specificcompound of Formula IX is 9-[2-(phosphonomethoxy)propyl]-guaninediphosphate; PMPGpp, or a pharmaceutically acceptable salt thereof. Aspecific compound of Formula IX is(R)-9-[2-(phosphonomethoxy)propyl]-guanine diphosphate; (R)-PMPGpp, or apharmaceutically acceptable salt thereof. A specific compound of FormulaIX is (S)-9-[2-(phosphonomethoxy)propyl]-guanine diphosphate;(S)-PMPGpp, or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula IX is the enriched orisolated (R) enantiomer. In another embodiment, the compound of FormulaIX is enriched or isolated (S) enantiomer.

In accordance with this invention, methods are provided for inhibitingtelomere elongation comprising contacting a cell with the compounds ofFormula IX of this invention or the pharmaceutical compositions of thisinvention. In some embodiments, the cell is a cancer cell.

In accordance with this invention, methods are provided for shorteningtelomere length in a cell or tissue comprising contacting the cell ortissue with the compounds of Formula VIII of this invention or thepharmaceutical compositions of this invention.

In accordance with this invention, methods are provided for treatingcancer in a patient by administering an effective amount of thecompounds of Formula IX of this invention or the pharmaceuticalcompositions of this invention to the patient. In one embodiment, thecancer is metastatic cancer. In one embodiment, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma).

In accordance with this invention, methods are provided for treating apatient by administering an effective amount of the compounds of FormulaIX of this invention or the pharmaceutical compositions of thisinvention wherein the method involves oral, intra-arterial, intranasal,intraperitoneal, intravenous, intramuscular, subcutaneous, ortransdermal administration of the compound or the pharmaceuticalcompositions of the invention.

Use of the compounds of Formula VIII in medicine.

Use of the compounds of Formula VIII and Formula IX for treating cancer.

The compounds of the invention inhibit the elongation or extension oftelomeres in cells by telomerase, including cancer cells, the resultanteffect of which is to inhibit proliferation of the cells. Accordingly, aprimary application of the compounds of the invention is as cancertherapeutics, and the invention provides pharmaceutical formulations ofthe compounds that may be utilized in this manner.

Compounds of the invention, including these exemplary compounds, areshown to have superior cellular uptake properties, compared tocorresponding unmodified nucleotides, and therefore to be more effectiveinhibitors of telomere elongation. As a consequence of these properties,compounds of the invention are highly effective inhibitors of cancercell proliferation.

It has been found that the compounds of the present invention act assubstrates for the telomerase enzyme and successfully compete with dGTPfor the nucleotide binding site of the telomerase enzyme. The compoundsdo not inhibit the activity of the telomerase enzyme. Rather thecompounds are incorporated into the telomere by the telomerase enzymethereby terminating the telomere strand. Once incorporated, thetelomerase enzyme is unable to attach further naturally occurring dNTPs,such as dGTP or dTTP to the telomere. In this manner, the elongation oftelomeres of chromosomes in cells expressing telomerase is halted orinhibited. Failure of the cells to lengthen their telomeres will put thecells into crisis or apoptosis.

The compounds of the present invention may be used in methods to inhibittelomere elongation. Such methods comprise contacting a cell or tissuewith a compound of the invention.

The compounds of the present invention may also be used to inhibitelongation of telomeres in cells that express telomerase, therebyinhibiting the proliferation of such cells. Such methods comprisecontacting a cell or cells having telomerase activity with a compound ofthe invention. Cells treated in this manner, which may be cells invitro, or cells in vivo, or cells ex vivo, will generally undergotelomere shortening and cease proliferating. Since cancer cells requiretelomere elongation for long-term proliferation, the compounds of theinvention are particularly useful for inhibiting the growth of cancercells, and may be used in therapeutic applications to treat cancer.

Aspects of the invention therefore include the compounds as describedherein for use in medicine, and in particular for use in treatingcancer.

Also provided herein are pharmaceutical compositions comprising acompound according to the invention formulated with a pharmaceuticallyacceptable excipient

In other aspects, provided herein are compounds comprising formula(VIII),

-   -   wherein G is selected from guanine-9-yl, or its 1-deaza or        3-deaza analogs, Y independently is —OH,        —NH(CH₂)_(n)NH(CH₂)_(n)NHR3; or —N[(CH2)nNH2](CH2)nNHR³; R³ is        —H or —(CH₂)_(n)NH₂; n independently is 2-4; with the proviso        that at least one Y is —NH(CH₂)_(n)NH(CH₂)nNHR³; or        —N[(CH₂)_(n)NH₂](CH₂)_(n)NHR³; and the salts, hydrates,        tautomers and solvates thereof. In some embodiments, G is        guanine-9-yl. In some embodiment at least one Y is        —NH(CH₂)_(n)NH(CH₂)_(n)NHR³, R³ is —H or —(CH₂)_(n)NH₂ and n        independently is 2-4. In some embodiments, at least one Y is        —NH(CH₂)₃NH(CH₂)₄NH₂ or —NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂. In some        embodiments at least one Y is —N[(CH₂)_(n)NH₂](CH₂)_(n)NHR₃, R₃        is —H or —(CH₂)_(n)NH₂ and n independently is 2-4. In some        embodiments, the compound of formula VIII is the enriched or        isolated (R) enantiomer. In some embodiments, the compound of        formula VIII is the enriched or isolated (S) enantiomer.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds disclosed herein with a pharmaceuticallyacceptable excipient.

In yet other aspects, provided herein are methods for inhibitingtelomere elongation comprising contacting a cell with any of thecompounds or the pharmaceutical compositions disclosed herein. In someembodiments, the cell is a cancer cell.

In still other aspects, provided herein are methods for shorteningtelomere length in a cell or tissue comprising contacting the cell ortissue with any of the compounds described herein or any of thepharmaceutical composition described herein.

In other aspects, provided herein are methods for treating cancer in apatient by administering an effective amount of any of the compoundsdescribed herein or any of the pharmaceutical composition describedherein to the patient. In some embodiments, the cancer is metastaticcancer. In some embodiments, the cancer is a cancer of the skin,connective tissue, adipose, breast, lung, liver, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, colorectal, prostate,central nervous system (CNS), brain, retina and hematologic tumors (suchas myeloma, leukemia and lymphoma). In some embodiments, of any of theembodiments disclosed herein, the method involves oral, intra-arterial,intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous,or transdermal administration.

In other aspects, provided herein are methods for inhibiting telomereelongation comprising contacting a cell with a compound of formula (IX)

-   -   wherein G is selected from guanine-9-yl, or its 1-deaza or        3-deaza analogs, X independently is —OH, a monphosphate, a        diphosphate or —OCH(R¹)OC(O)OR¹, R¹ independently is —H, or        C₁-C₅ alkyl; and the salts, hydrates, tautomers and solvates        thereof; under conditions wherein telomere elongation is        inhibited. In some embodiments, at least one X is —OCH₂OC(O)OR₁        and R₁ is C₁-C₅ alkyl. In some embodiments, one X is —OH and the        other X is —OCH₂OC(O)OR¹ and R₁ is C₁-C₅ alkyl. In some        embodiments, the compound of formula IX is        9-[2-(phosphonomethoxy)propyl]-guanine diphosphate, or a        pharmaceutically acceptable salt thereof. In some embodiments        the compound of formula IX is the enriched or isolated (R)        enantiomer. In some embodiments, the compound of formula IX is        enriched or isolated (S) enantiomer. In some embodiments of any        of the embodiments provided herein, the cell is a cancer cell.        In some embodiments, the cancer is metastatic cancer. In some        embodiments, the cancer is a cancer of the skin, connective        tissue, adipose, breast, lung, liver, stomach, pancreas, ovary,        cervix, uterus, kidney, bladder, colon, colorectal, prostate,        central nervous system (CNS), brain, retina and hematologic        tumors (such as myeloma, leukemia and lymphoma). In some        embodiments of any of the embodiments provided herein, the        method involves oral, intra-arterial, intranasal,        intraperitoneal, intravenous, intramuscular, subcutaneous, or        transdermal administration.

In some aspects, provided herein are methods for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds disclosed herein.

In other aspects, provided herein are methods for treating cancer in apatient by administering an effective amount of the compound of any ofthe compounds disclosed herein to the patient.

In other aspects, provided herein are uses of any of the compoundsprovided herein in medicine.

In other aspects, provided herein are uses of any of the compoundsprovided herein for treating cancer.

In other aspects, provided herein are compounds comprising formula(VIII),

wherein

R¹ and R² are independently selected from —NR^(1a)R^(1b) and OR^(1c);wherein

-   -   R^(1a) and R^(1b) are independently selected from hydrogen,        optionally substituted C₁₋₂₀alkyl, optionally substituted        polyamine, and —CH(R^(1d))—C(O)OR^(1e), wherein        -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,            aryl, substituted aryl, arylalkyl, substituted arylalkyl,            heteroalkyl, substituted heteroalkyl, heteroaryl,            substituted heteroaryl, heteroarylalkyl and substituted            heteroarylalkyl; and        -   R^(1e) is hydrogen or C₁₋₆alkyl;    -   R^(1c) is selected from hydrogen, alkyl, and aryl;

wherein at least one of R¹ and R² is —NR^(1a)R^(1b);

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃,or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH. —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, R¹ is different from R². In some embodiments, oneof R¹ and R² carries a positive charge and the other carries a negativecharge. In some embodiments, the compound of formula (I) is the enrichedor isolated (R) enantiomer at the stereocenter bearing R⁴.

In some embodiments, R¹ is —NR^(1a)R^(1b) and R² is OR^(1c). In someembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. Insome embodiments, one of R^(1a) and R^(1b) is a polyamine; and R² is OH.In some embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In someembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); whereinR^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from2 to 4. In some embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In some embodiments, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, heteroaryl, andsubstituted heteroaryl. In some embodiments, wherein R^(1d) is apositively charged amino acid side chain. In some embodiments, wherein—CH(R^(1d))—C(O)OR^(1e) is an amino acid selected from lysine, arginine,and histidine.

In some embodiments, R^(3a) and R^(3b) are hydrogen. In someembodiments, one of R^(3a) and R^(3b) is halo. In some embodiments, W isO. In some embodiments, R⁴ is —OH. In some embodiments, R⁴ is selectedfrom —NH₂ and N₃. In some embodiments, R⁴ is —CH═CH₂. In someembodiments, R⁴ is C₁₋₂ alkyl. In some embodiments, R⁴ is optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, orN₃. In some embodiments, R^(5a) and R^(5b) are hydrogen. In someembodiments, R⁷ is hydrogen. In some embodiments, R⁷ is fluoro. In someembodiments, X is O.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds of Formula (I) with a pharmaceuticallyacceptable excipient.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with any of the compounds ofFormula (I) or pharmaceutical compositions comprising a compound ofFormula (I). In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds of Formula (I) or pharmaceutical compositionscomprising a compound of Formula (I).

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds of Formula (I) or pharmaceuticalcompositions comprising a compound of Formula (I). In some embodiments,the cell proliferative disorder is cancer. In some embodiments, thecancer is metastatic cancer. In some embodiments, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma). In some embodiments,the compound or pharmaceutical composition is administered orally,intra-arterially, intranasally, intraperitoneally, intravenously,intramuscularly, subcutaneously, or transdermally.

In other aspects, provided herein is a compound of formula (II):

wherein

X¹ is NH or O;

X² is NH or O;

R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl, optionallysubstituted C₁₋₂₀alkenyl, or optionally substituted C₁₋₂₀alkynyl;

R¹ is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

-   -   R^(1a) and R^(1b) are independently selected from hydrogen,        optionally substituted C₁₋₂₀alkyl, optionally substituted        polyamine, and —CH(R^(1d))—C(O)OR^(1e), wherein        -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,            aryl, substituted aryl, arylalkyl, substituted arylalkyl,            heteroalkyl, substituted heteroalkyl, heteroaryl,            substituted heteroaryl, heteroarylalkyl, and substituted            heteroarylalkyl; and        -   R^(1e) is hydrogen or C₁₋₆alkyl;    -   R^(1c) is selected from hydrogen, alkyl, and aryl:

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃,or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen:

R⁷ is hydrogen or fluoro; and

X³ is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, X¹ and X² are O. In some embodiments, wherein R³⁰is C₁₋₂₀alkyl. In some embodiments, R¹ carries a positive charge or anegative charge. In some embodiments, the compound of formula (II) isthe enriched or isolated (R) enantiomer at the stereocenter bearing R⁴.

In some embodiments, R¹ is —NR^(1a)R^(1b). In some embodiments, one ofR^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. In some embodiments, oneof R^(1a) and R^(1b) is a polyamine; and R² is OH. In some embodiments,one of R^(1a) and R^(1b) is —(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x)is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from 2 to4. In some embodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x);wherein R^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently anumber from 2 to 4. In some embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In some embodiments, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, heteroaryl, andsubstituted heteroaryl. In some embodiments, R^(1d) is a positivelycharged amino acid side chain. In some embodiments,—CH(R^(1d))—C(O)OR^(1e) is an amino acid selected from lysine, arginine,and histidine.

In some embodiments, R^(3a) and R^(3b) are hydrogen. In someembodiments, one of R^(3a) and R^(3b) is halo. In some embodiments, W isO. In some embodiments, R⁴ is —OH. In some embodiments, R⁴ is selectedfrom —NH₂ and N₃. In some embodiments, R⁴ is —CH═CH₂. In someembodiments, R⁴ is C₁₋₂ alkyl. In some embodiments, R⁴ is optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, orN₃. In some embodiments, R^(5a) and R^(5b) are hydrogen. In someembodiments, R⁷ is hydrogen. In some embodiments, R⁷ is fluoro. In someembodiments, X is O.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds of Formula (II) with a pharmaceuticallyacceptable excipient.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with any of the compounds ofFormula (II) or pharmaceutical compositions comprising a compound ofFormula (II). In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds of Formula (II) or pharmaceutical compositionscomprising a compound of Formula (II).

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds of Formula (II) or pharmaceuticalcompositions comprising a compound of Formula (II). In some embodiments,the cell proliferative disorder is cancer. In some embodiments, thecancer is metastatic cancer. In some embodiments, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma). In some embodiments,the compound or pharmaceutical composition is administered orally,intra-arterially, intranasally, intraperitoneally, intravenously,intramuscularly, subcutaneously, or transdermally.

In other aspects, provided herein is a compound of formula (III):

wherein

R¹ and R² are independently selected from —NR^(1a)R^(1b) and OR^(1c);wherein

-   -   R^(1a) and R^(1b) are independently selected from hydrogen,        optionally substituted C₁₋₂₀alkyl, optionally substituted        polyamine, and —CH(R^(1d))—C(O)OR^(1e), wherein        -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,            aryl, substituted aryl, arylalkyl, substituted arylalkyl,            heteroalkyl, substituted heteroalkyl, heteroaryl,            substituted heteroaryl, heteroarylalkyl, and substituted            heteroarylalkyl; and        -   R^(1e) is hydrogen or C₁₋₆alkyl;    -   R^(1c) is selected from hydrogen, alkyl, and aryl;

wherein at least one of R¹ and R² is —NR^(1a)R^(1b);

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(6a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, R¹ is different from R². In some embodiments, oneof R¹ and R² carries a positive charge and the other carries a negativecharge.

In some embodiments, R¹ is —NR^(1a)R^(1b) and R² is OR^(1c). In someembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. Insome embodiments, one of R^(1a) and R^(1b) is a polyamine; and R² is OH.In some embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In someembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); whereinR^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from2 to 4. In some embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In some embodiments, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, heteroaryl, andsubstituted heteroaryl. In some embodiments, R^(1d) is a positivelycharged amino acid side chain. In some embodiments,—CH(R^(1d))—C(O)OR^(1e) is an amino acid selected from lysine, arginine,and histidine.

In some embodiments, R^(3a) and R^(3b) are hydrogen. In someembodiments, one of R^(3a) and R^(3b) is halo. In some embodiments, W isO. In some embodiments, R^(4a) and R^(4b) are hydrogen. In someembodiments, one of R^(5a) and R^(5b) is —OH. In some embodiments, oneof R^(5a) and R^(5b) is selected from —NH₂ and N₃. In some embodiments,one of R^(5a) and R^(5b) is —CH═CH₂. In some embodiments, one of R^(5a)and R^(5b) is C₁₋₂ alkyl. In some embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In some embodiments, R^(6a) and R^(6b) are hydrogen.In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is fluoro.In some embodiments, X is O.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds of Formula (III) with a pharmaceuticallyacceptable excipient.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with any of the compounds ofFormula (III) or pharmaceutical compositions comprising a compound ofFormula (III). In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds of Formula (III) or pharmaceutical compositionscomprising a compound of Formula (III).

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds of Formula (III) or pharmaceuticalcompositions comprising a compound of Formula (III). In someembodiments, the cell proliferative disorder is cancer. In someembodiments, the cancer is metastatic cancer. In some embodiments, thecancer is a cancer of the skin, connective tissue, adipose, breast,lung, liver, stomach, pancreas, ovary, cervix, uterus, kidney, bladder,colon, colorectal, prostate, central nervous system (CNS), brain, retinaand hematologic tumors (such as myeloma, leukemia and lymphoma). In someembodiments, the compound or pharmaceutical composition is administeredorally, intra-arterially, intranasally, intraperitoneally,intravenously, intramuscularly, subcutaneously, or transdermally.

In other aspects, provided herein is a compound of formula (IV):

wherein

X¹ is NH or O;

X² is NH or O;

R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl, optionallysubstituted C₁₋₂₀ alkenyl, or optionally substituted C₁₋₂₀alkynyl;

R¹ is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

-   -   R^(1a) and R^(1b) are independently selected from hydrogen,        optionally substituted C₁₋₂₀alkyl, optionally substituted        polyamine, and —CH(R^(1d))—C(O)OR^(1e), wherein        -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,            aryl, substituted aryl, arylalkyl, substituted arylalkyl,            heteroalkyl, substituted heteroalkyl, heteroaryl,            substituted heteroaryl, heteroarylalkyl, and substituted            heteroarylalkyl; and        -   R^(1e) is hydrogen or C₁₋₆alkyl;    -   R^(1c) is selected from hydrogen, alkyl, and aryl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂₀ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen:

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen:

R^(6a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X³ is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, X¹ and X² are O. In some embodiments, R³⁰ isC₁₋₂₀alkyl. In some embodiments, R¹ carries a positive charge or anegative charge.

In some embodiments, R¹ is —NR^(1a)R^(1b). In some embodiments, one ofR^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. In some embodiments, oneof R^(1a) and R^(1b) is a polyamine; and R² is OH. In some embodiments,one of R^(1a) and R^(1b) is —(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x)is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from 2 to4. In some embodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x);wherein R^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently anumber from 2 to 4. In some embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In some embodiments, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, heteroaryl, andsubstituted heteroaryl. In some embodiments, R^(1d) is a positivelycharged amino acid side chain. In some embodiments,—CH(R^(1d))—C(O)OR^(1e) is an amino acid selected from lysine, arginine,and histidine.

In some embodiments, R^(3a) and R^(3b) are hydrogen. In someembodiments, one of R^(3a) and R^(3b) is halo. In some embodiments, W isO. In some embodiments, R^(4a) and R^(4b) are hydrogen. In someembodiments, one of R^(5a) and R^(5b) is —OH. In some embodiments, oneof R^(5a) and R^(5b) is selected from —NH₂ and N₃. In some embodiments,one of R^(5a) and R^(5b) is —CH═CH₂. In some embodiments, one of R^(5a)and R^(5b) is C₁₋₂ alkyl. In some embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In some embodiments, R^(6a) and R^(6b) are hydrogen.In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is fluoro.In some embodiments, X is O.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds of Formula (IV) with a pharmaceuticallyacceptable excipient.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with any of the compounds ofFormula (IV) or pharmaceutical compositions comprising a compound ofFormula (IV). In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds of Formula (IV) or pharmaceutical compositionscomprising a compound of Formula (IV).

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds of Formula (IV) or pharmaceuticalcompositions comprising a compound of Formula (IV). In some embodiments,the cell proliferative disorder is cancer. In some embodiments, thecancer is metastatic cancer. In some embodiments, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma). In some embodiments,the compound or pharmaceutical composition is administered orally,intra-arterially, intranasally, intraperitoneally, intravenously,intramuscularly, subcutaneously, or transdermally.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with a compound of formula (V):

wherein

R¹ and R² are independently selected from —OH, monophosphate,diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) areindependently selected from hydrogen and C₁₋₅alkyl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH:

R⁴ is selected from —OH, —NH₂, N₃, —CH═CH₂, and optionally substitutedC₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen. —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue witha compound of formula (V).

wherein

R¹ and R² are independently selected from —OH, monophosphate,diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) areindependently selected from hydrogen and C₁₋₅alkyl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from —OH, —NH₂, N₃, —CH═CH₂, and optionally substitutedC₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen:

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of a compound of formula (VI).

wherein

R¹ and R² are independently selected from —OH, monophosphate,diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) areindependently selected from hydrogen and C₁₋₅alkyl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH. —NH₂, N₃,or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen. —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen;

R^(6a) and R^(6b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen:

R⁷ is hydrogen or fluoro; and

X is O, S, or NH:

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, the cell proliferative disorder is cancer. In someembodiments, the cancer is metastatic cancer. In some embodiments, thecancer is a cancer of the skin, connective tissue, adipose, breast,lung, liver, stomach, pancreas, ovary, cervix, uterus, kidney, bladder,colon, colorectal, prostate, central nervous system (CNS), brain, retinaand hematologic tumors (such as myeloma, leukemia and lymphoma). In someembodiments, the compound or pharmaceutical composition is administeredorally, intra-arterially, intranasally, intraperitoneally,intravenously, intramuscularly, subcutaneously, or transdermally. Insome embodiments, the cell proliferative disorder is cancer. In someembodiments, the cancer is metastatic cancer.

In other aspects, provided herein is a compound of Formula (VII):

wherein

R² is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl;

R^(1c) is selected from hydrogen, alkyl, and aryl:

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen. —OH, —NH₁,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃. —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen;

R^(6a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂.N₃. —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

L¹ is an optionally substituted polyamine;

m is zero or one;

n is zero or one;

and salts, hydrates, solvates, and tautomers, thereof.

In some embodiments, L¹ is a polyamine.

In some embodiments, R² is —NR^(1a)R^(1b) and R² is OR^(1c). In someembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. Insome embodiments, one of R^(1a) and R^(1b) is a polyamine; and R² is OH.In some embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In someembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); whereinR^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from2 to 4. In some embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In some embodiments, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, heteroaryl, andsubstituted heteroaryl. In some embodiments, R^(1d) is a positivelycharged amino acid side chain. In some embodiments,—CH(R^(1d))—C(O)OR^(1e) is an amino acid selected from lysine, arginine,and histidine.

In some embodiments, R^(3a) and R^(3b) are hydrogen. In someembodiments, one of R^(3a) and R^(3b) is halo. In some embodiments, W isO. In some embodiments, R^(4a) and R^(4b) are hydrogen. In someembodiments, one of R^(5a) and R^(5b) is —OH. In some embodiments, oneof R^(5a) and R^(5b) is selected from —NH₂ and N₃. In some embodiments,one of R^(5a) and R^(5b) is —CH═CH₂. In some embodiments, one of R^(5a)and R^(5b) is C₁₋₂ alkyl. In some embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In some embodiments, R^(6a) and R^(6b) are hydrogen.In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is fluoro.In some embodiments, X is O.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the compounds of Formula (VII) with a pharmaceuticallyacceptable excipient.

In other aspects, provided herein is a method for inhibiting telomereelongation comprising contacting a cell with any of the compounds ofFormula (VII) or pharmaceutical compositions comprising a compound ofFormula (VII). In some embodiments, the cell is a cancer cell.

In other aspects, provided herein is a method for shortening telomerelength in a cell or tissue comprising contacting the cell or tissue withany of the compounds of Formula (VII) or pharmaceutical compositionscomprising a compound of Formula (VII).

In other aspects, provided herein is a method of treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds of Formula (VII) or pharmaceuticalcompositions comprising a compound of Formula (VII). In someembodiments, the cell proliferative disorder is cancer. In someembodiments, the cancer is metastatic cancer. In some embodiments, thecancer is a cancer of the skin, connective tissue, adipose, breast,lung, liver, stomach, pancreas, ovary, cervix, uterus, kidney, bladder,colon, colorectal, prostate, central nervous system (CNS), brain, retinaand hematologic tumors (such as myeloma, leukemia and lymphoma). In someembodiments, the compound or pharmaceutical composition is administeredorally, intra-arterially, intranasally, intraperitoneally,intravenously, intramuscularly, subcutaneously, or transdermally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a 15% polyacrylamide gel containing 7 M ureashowing the primer extension products in the presence of compounds.Reactions containing 50 or 100 μM dGTP, (lanes 1 and 2) show acharacteristic 6 nucleotide ladder. Lanes 8-11 contain increasingconcentrations of PMPGpp (ID#142692) in addition to 50 μM dGTP. Lane 12contains only 50 μM PMPGpp (ID#142692). Lanes 3-7 show a comparisonusing the known chain terminator, 3′-azido-dGTP.

FIG. 2A shows U87 gb cells were treated with PMPG diisopropyloxy ester(ID#142715) (lane 4), 2′ deoxy, 3′ C6 phenyl-amide, 5′thiophosphateguanosine (lane 3), 2′ deoxy, 3′ azido, 5′ thiophosphate gunosine (lane2) or untreated (lane 1).

FIG. 2B shows Caki 1 cells treated five weeks with either 10 μM or 20 μMPMPG diisopropyloxy ester (ID#142715) (lanes 3 and 4) as compared withuntreated or DMSO treated cells (lanes 1 and 2).

FIG. 2C shows the treatment of A549 cells for seven weeks using either10 μM or 20 μM PMPG diisopropyloxy ester (ID#142715) (lanes 3 and 4) ascompared with untreated or DMSO treated cells (lanes 1 and 2).

FIG. 3A shows the growth curves of U87 glioblastoma cells treated witheither 10 μM or 20 μM PMPG diisopropyloxy ester (ID#142715) for 47 days.

FIG. 3B shows U87 glioblastoma cells that were treated with either 10 μMor 20 μM PMPG diisopropyloxy ester (ID#142715) for five weeks thenseeded in equal cell numbers (75,000/well). Photos were taken 6 daysafter seeding.

FIG. 4A is a photograph of a resulting gel from a primer extension assayrun in competition mode. From left to right, Lanes 1 and 2 (marked “−”)show a characteristic 6 nucleotide ladder. Lanes 3-8 contain increasingconcentrations of (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diphosphate (R-PMPGpp) (ID#142692), from 0.25 μM to 50 μM.

FIG. 4B shows (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic aciddiphosphate (R-PMPGpp) (ID#142692) telomerase inhibition activity. Thepercent activity was determined based on values obtained byPhosphoImager analysis of the intensity of the gel bands in FIG. 4A.

FIG. 5 shows an Ex Vivo TRAP assay with PMPG prodrug (ID#142715). Twoindependent assay results are shown for the same compound.

FIG. 6 shows the growth curves for Caki-1 cells treated with 10 μM or 20μM PMPG prodrug [R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diisopropyloxy ester](ID#142715).

FIG. 7 shows the growth curves for A549 cells treated with 10 μM or 20μM PMPG prodrug [R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diisopropyloxy ester](ID#1427150) compared to 5 μM or 10 μMImetelstat (163 L).

FIG. 8A is a photograph of a resulting gel from a primer extensionassay. From left to right, Lane 1 (marked “−”) shows a characteristic 6nucleotide ladder. Lanes 2-5 contain increasing concentrations of(R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate(R-PMPGpp) (ID#142692), from 0.4 μM to 50 μM. Lanes 6-9 contain from 0.4μM to 50 μM of PMIBeGpp (ID#142810).

FIG. 8B shows the dose response telomerase inhibition for R-PMPGpp(ID#142692) as compared to PMIBeGpp (ID#142810). The percent activitywas determined based on values obtained by PhosphoImager analysis of theintensity of the gel bands in FIG. 8A.

FIG. 9 shows the growth curves for Caki-1 cells treated with 10 or 20 μMPMIBeG diisopropyloxy ester (ID#142820) or PMPG free acid (ID#142693).

FIG. 10 shows the gel resulting from a telomere repeat fragment assayusing A549 cells. Lane 1 is a ladder from 21.2 kb to 1.9 kb. Lanes 2 and3 have 10 μM and 5 μM, respectively, of PMPG free acid (ID#142693).Lanes 4 to 6 have 10 μM, 5 μM, and 2.5 μM, respectively, of PMPG prodrug(ID#142715). Lane 7 has 2 μM of Imetelstat (163 L). Lane 8 has 2 μM of amismatch. Lanes 9 and 10 have 20 μM and 10 μM, respectively, of PMIBeGfree acid[(((2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methyl)allyl)oxy)methyl)phosphonicacid] (ID#142811). Indicated amounts of each compound were added to A549cells. After approximately 70 population doublings, the cells wereharvested and telomere lengths determined using TRF.

FIG. 11 shows the plot of fitted growth curves (Imetelstat Day<41)obtained when low-passage Caki-1 cells were injected subcutaneously intothe flank of SCID hairless outbred (SHO) mice receiving 30 mg/kg ofvehicle (PBS with 0.4% Tween 20), Imetelstat, PMPG free acid (ID#142693)or PMPG prodrug (ID#142715) via intraperitoneal injection.

DETAILED DESCRIPTION

This invention relates to, inter alia, compounds useful for inhibitingtelomere elongation. More specifically, the invention providesnucleotide analogs that are incorporated into telomeres by telomerasethereby inhibiting elongation of telomeres. The compounds are useful intreating cancer and other cell proliferative diseases.

I. General Techniques

The practice of the invention will employ, unless otherwise indicated,conventional techniques in nucleic acid chemistry, molecular biology,microbiology, cell biology, biochemistry, and immunology, which are wellknown to those skilled in the art. Such techniques are explained fullyin the literature, such as, Molecular Cloning: A Laboratory Manual,second edition (Sambrook et al., 1989) and Molecular Cloning: ALaboratory Manual, third edition (Sambrook and Russel, 2001), (jointlyreferred to herein as “Sambrook”); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987, including supplements through2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994).Nucleic acids can be synthesized in vitro by well-known chemicalsynthesis techniques, as described in, e.g., Carruthers (1982) ColdSpring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem.Soc. 105:661; Belousov (1997) Nucleic Acids Res. 5 25:3440-3444; Frenkel(1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth.Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; Komberg andBaker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992); Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlmann and Peyman,Chemical Reviews, 90:543-584, 1990.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, 4^(th) edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5^(th) edition, Wiley-Interscience, 2001.

II. Definitions

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

The term “saturated,” as used herein, pertains to compounds and/orgroups which do not have any carbon-carbon double bonds or carbon-carbontriple bonds.

The term “unsaturated,” as used herein, pertains to compounds and/orgroups which have at least one carbon-carbon double bond orcarbon-carbon triple bond.

The term “cyclic,” as used herein, pertains to compounds and/or groupswhich have one ring, or two or more rings (e.g., spiro, fused, bridged).

The term “ring,” as used herein, pertains to a closed ring of from 3 to10 covalently linked atoms, more preferably 3 to 8 covalently linkedatoms.

The phrase “optionally substituted,” as used herein, pertains to aparent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted,” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, appended to, or ifappropriate, fused to, a parent group.

The substituents are described in more detail below.

C₁₋₇alkyl: The term “C₁₋₇alkyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from aC₁₋₇hydrocarbon compound having from 1 to 7 carbon atoms, which may bealiphatic or alicyclic, or a combination thereof, and which may besaturated, partially unsaturated, or fully unsaturated. The term “C₂₋₄alkyl” (and similar terms), as used herein, pertains to analogousmoieties having from 2 to 4 carbon atoms. Examples are —CH₂CH₃;—CH₂CH₂CH₃; —CH(CH₃)₂; —CH₂CH₂CH₂CH₃; —CH₂CH(CH₃)₂; —CH(CH₃)CH₂CH₃;—C(CH₃)₃; —CH₂CH₂CH₂CH₂CH₃; —CH₂CH₂CH(CH₃)₂; —CH₂CH(CH₃)CH₂CH₃; —CH(CH₃)CH₂CH₂CH₃; —CH(CH₂CH₃)₂; —C(CH₃)₂CH₂CH₃; —CH(CH₃) CH(CH₃)₂; —CH₂C(CH₃)₃;cyclopropyl; cyclobutyl; cyclopropymethyl; cyclobutylmethyl;1-cyclopropyl-1-ethyl; 2-cyclopropyl-1-ethyl; and cyclopentyl.

C_(1-n)alkyl: The term “C_(1-n)alkyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from aC_(1-n)hydrocarbon compound having from 1 to n carbon atoms, which maybe aliphatic or alicyclic, or a combination thereof, and which may besaturated, partially unsaturated, or fully unsaturated, wherein n is anumber greater than one. Likewise, C₁₋₂₀alkyl: The term “C₁₋₂₀alkyl,” asused herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a C₁₋₂₀hydrocarbon compound having from 1 to 20carbon atoms, which may be aliphatic or alicyclic, or a combinationthereof, and which may be saturated, partially unsaturated, or fullyunsaturated.

Examples of (unsubstituted) saturated linear C₁₋₇alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl(amyl).

Examples of (unsubstituted) saturated branched C₁₋₇alkyl groups include,but are not limited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl,and neo-pentyl.

Examples of saturated alicyclic (carbocyclic) C₁₋₇alkyl groups (alsoreferred to as “C₃₋₇cycloalkyl” groups) include, but are not limited to,unsubstituted groups such as cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl, as well as substituted groups (e.g., groups which comprisesuch groups), such as methylcyclopropyl, dimethylcyclopropyl,methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl,dimethylcyclopentyl, methylcyclohexyl, dimethylcyclohexyl,cyclopropylmethyl and cyclohexylmethyl.

Examples of (unsubstituted) unsaturated C₁₋₇alkyl groups which have oneor more carbon-carbon double bonds (also referred to as “C₂₋₇alkenyl”groups) include, but are not limited to, ethenyl (vinyl, —CH═CH₂),2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂), butenyl,pentenyl, and hexenyl.

Examples of (unsubstituted) unsaturated C₁₋₇alkyl groups which have oneor more carbon-carbon triple bonds (also referred to as “C₂₋₇alkynyl”groups) include, but are not limited to, ethynyl (ethinyl) and2-propynyl (propargyl).

Examples of unsaturated alicyclic (carbocyclic) C₁₋₇alkyl groups whichhave one or more carbon-carbon double bonds (also referred to as“C₃₋₇cycloalkenyl” groups) include, but are not limited to,unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl,and cyclohexenyl, as well as substituted groups (e.g., groups whichcomprise such groups) such as cyclopropenylmethyl andcyclohexenylmethyl.

C₃₋₂₀heterocyclyl: The term “C₃₋₂₀heterocyclyl,” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a C₃₋₂₀heterocyclic compound, said compound havingone ring, or two or more rings (e.g., spiro, fused, bridged), and havingfrom 3 to 20 ring atoms, atoms, of which from 1 to 10 are ringheteroatoms, and wherein at least one of said ring(s) is a heterocyclicring. Preferably, each ring has from 3 to 7 ring atoms, of which from 1to 4 are ring heteroatoms. “C₃₋₂₀” denotes ring atoms, whether carbonatoms or heteroatoms.

Examples of (non-aromatic) monocyclic heterocyclyl groups include, butare not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane(tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane(C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

O₃: trioxane (C₆);

N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);

N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);

N₂O₁: oxadiazine (C₆);

O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N₁O₁S₁: oxathiazine (C₆).

Examples of substituted (non-aromatic) monocyclic heterocyclyl groupsinclude saccharides, in cyclic form, for example, furanoses (C₅), suchas arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, andpyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

Examples of heterocyclyl groups which are also heteroaryl groups aredescribed below with aryl groups.

C₅₋₂₀aryl: The term “C₅₋₂₀aryl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of a C₅₋₂₀aromatic compound, said compound having one ring, ortwo or more rings (e.g., fused), and having from 5 to 20 ring atoms, andwherein at least one of said ring(s) is an aromatic ring. Preferably,each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups,” inwhich case the group may conveniently be referred to as a“C₅₋₂₀carboaryl” group.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e., phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indene (C₉), isoindene (C₉), and fluorene (C₁₃).

Alternatively, the ring atoms may include one or more heteroatoms,including but not limited to oxygen, nitrogen, and sulfur, as in“heteroaryl groups.” In this case, the group may conveniently bereferred to as a “C₅₋₂₀heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of monocyclic heteroaryl groups include, but are not limitedto, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅); N₂: imidazole (1,3-diazole) (C₅),pyrazole (1,2-diazole) (C₅), pyridazine (1,2-diazine) (C₆), pyrimidine(1,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine(1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and, N₄: tetrazole (C₅).

The above C₁₋₇alkyl, C₃₋₂₀heterocyclyl, and C₃₋₂₀aryl groups, whetheralone or part of another substituent, may themselves optionally besubstituted with one or more groups selected from themselves and theadditional substituents listed below.

Hydrogen: —H. Note that if the substituent at a particular position ishydrogen, it may be convenient to refer to the compound as being“unsubstituted” at that position.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇alkylgroup (also referred to as a C₁₋₇alkoxy group, discussed below), aC₃₋₂₀heterocyclyl group (also referred to as a C₃₋₂₀heterocyclyloxygroup), or a C₅₋₂₀aryl group (also referred to as a C₅₋₂₀aryloxy group),preferably a C₁₋₇alkyl group.

C₁₋₇alkoxy: —OR, wherein R is a C₁₋₇alkyl group. Examples of C₁₋₇alkoxygroups include, but are not limited to, —OCH₃ (methoxy), —OCH₂CH₃(ethoxy) and —OC(CH₃)₃ (tert-butoxy).

Oxo (keto, -one): ═O.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀arylacyl), preferably aC₁₋₇alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —COOH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C.sub.1-7alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxygroups include, but are not limited to, —OC(═O)CH₃ (acetoxy),—OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarbonyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR₁R₂,wherein R₁ and R₂ are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R₁ and R₂, togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Acylamido (acylamino): —NR₁C(═O)R₂, wherein R₁ is an amide substituent,for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or aC₅₋₂₀aryl group, preferably a C₁₋₇alkyl group, and R₂ is an acylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group,or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofacylamido groups include, but are not limited to, —NHC(═O)CH₃,—NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R₁ and R₂ may together form a cyclicstructure, as in, for example, succinimidyl, maleimidyl, andphthalimidyl, tetrazolyl: (a five membered aromatic ring having fournitrogen atoms and one carbon atom).

Amino: —NR₁R², wherein R₁ and R₂ are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R₁ and R₂, taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Examples of amino groups include, but are not limited to,—NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples ofcyclic amino groups include, but are not limited to, aziridino,azetidino, pyrrolidino, piperidino, piperazino, morpholino, andthiomorpholino.

Polyamine refers to polymers having an amine functionality in themonomer unit, either incorporated into the backbone, as inpolyalkyleneimines, or in a pendant group as in polyvinyl amines.

As mentioned above, a C₁₋₇alkyl group may be substituted with, forexample, hydroxy (also referred to as a C₁₋₇hydroxyalkyl group),C₁₋₇alkoxy (also referred to as a C₁₋₇alkoxyalkyl group), amino (alsoreferred to as a C₁₋₇aminoalkyl group), halo (also referred to as aC₁₋₇haloalkyl group), carboxy (also referred to as a C₁₋₇carboxyalkylgroup), and C₅₋₂₀aryl (also referred to as a C₅₋₂₀aryl-C₁₋₇alkyl group).

Similarly, a C₅₋₂₀aryl group may be substituted with, for example,hydroxy (also referred to as a C₅₋₂₀hydroxyaryl group), halo (alsoreferred to as a C₅₋₂₀haloaryl group), amino (also referred to as aC₅₋₂₀aminoaryl group, e.g., as in aniline), C₁₋₇alkyl (also referred toas a C₁₋₇alkyl-C₅₋₂₀aryl group, e.g., as in toluene), and C₁₋₇alkoxy(also referred to as a C₁₋₇alkoxy-C₅₋₂₀aryl group, e.g., as in anisole).

Included in the above are the well-known ionic, salt, solvate, andprotected forms of these substituents. For example, a reference tocarboxylic acid (—COOH) also includes the anionic (carboxylate) form(—COO⁻), a salt or solvate thereof, as well as conventional protectedforms. Similarly, a reference to an amino group includes the protonatedform (—N⁺HR¹R²), a salt or solvate of the amino group, for example, ahydrochloride salt, as well as conventional protected forms of an aminogroup. Similarly, a reference to a hydroxyl group also includes theanionic form (—O⁻), a salt or solvate thereof, as well as conventionalprotected forms of a hydroxyl group.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. In the case ofcancer or tumor, an effective amount of the drug may have the effect inreducing the number of cancer cells; reducing the tumor size; inhibiting(i.e., slow to some extent and preferably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent andpreferably stop) tumor metastasis; inhibiting, to some extent, tumorgrowth; and/or relieving to some extent one or more of the symptomsassociated with the disorder. An effective dosage can be administered inone or more administrations. For purposes of the present disclosure, aneffective dosage of drug, compound, or pharmaceutical composition is anamount sufficient to accomplish prophylactic or therapeutic treatmenteither directly or indirectly. As is understood in the clinical context,an effective dosage of a drug, compound, or pharmaceutical compositionmay or may not be achieved in conjunction with another drug, compound,or pharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including and preferably clinical results.For purposes of the present disclosure, beneficial or desired clinicalresults include, but are not limited to, one or more of the following:reducing the proliferation of (or destroying) cancerous cells,decreasing symptoms resulting from the disease, increasing the qualityof life of those suffering from the disease, decreasing the dose ofother medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of individuals.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

An “individual” or a “subject” or a “patient” is a mammal. Mammals alsoinclude, but are not limited to, farm animals, sport animals, pets (suchas cats, dogs, horses), primates, mice and rats. In some embodiments, anindividual is a human.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

It is understood that aspect and variations of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand variations.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsubcombination of chemical groups was individually and explicitlydisclosed herein.

III. Compounds

A. Formula I

The present disclosure provides a compound of Formula (I):

wherein

R¹ and R² are independently selected from —NR^(1a)R^(1b) and OR^(1c);wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl:

R^(1c) is selected from hydrogen, alkyl, and aryl;

wherein at least one of R¹ and R² is —NR^(1a)R^(1b);

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃,or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, R¹ is different from R². In certain embodiments,one of R¹ and R² carries a positive charge and the other carries anegative charge. In certain embodiments, the compound of Formula (I) isa zwitterion.

In certain embodiments, the compound of formula (I) is the enriched orisolated (R) enantiomer at the stereocenter bearing R⁴.

In Formula (I), R¹ and R² are independently selected from —NR^(1a)R^(1b)and OR^(1c); wherein at least one of R¹ and R² is —NR^(1a)R^(1b). Incertain embodiments, R¹ is —NR^(1a)R^(1b) and R² is OR^(1c). In certainembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH. Incertain embodiments, one of R^(1a) and R^(1b) is a polyamine; and R² isOH.

In certain embodiments, one of R^(1a) and R^(1b) is hydrogen. In certainembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl. In certaininstances, one of R^(1a) and R^(1b) is C₅₋₂₀alkyl, C₁₀₋₂₀alkyl,C₁₅₋₂₀alkyl, C₁₋₁₅alkyl, C₁₋₁₀alkyl, C₅₋₁₅alkyl, or C₅₋₁₅alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₈alkyl, C₉alkyl,C₁₀alkyl, C₁₁alkyl, or C₁₂alkyl.

In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In certainembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); whereinR^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from2 to 4. In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH₂, wherein n is a number from 2 to 4.

In certain embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In certain instances, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl. In certain instances, R^(1d) is selectedfrom hydrogen, alkyl, substituted alkyl, heteroaryl, and substitutedheteroaryl. In certain instances, R^(1d) is alkyl or substituted alkyl.

In certain instances, R^(1d) is an amino acid side chain. In certaininstances, —CH(R^(1d))—C(O)OR^(1e) is an amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. In certain instances, R^(1d) is a positively charged amino acidside chain. In certain instances, —CH(R^(1d))—C(O)OR^(1e) is an aminoacid selected from lysine, arginine, and histidine.

Amino acid refers to the natural (genetically encoded) or unnatural orsynthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “natural” refers to thegenetically encoded 20 amino acids in their natural configuration.“Unnatural amino acids” have been modified after protein synthesis,and/or have a chemical structure in their side chain(s) different fromthat of the standard amino acids. Unnatural amino acids can bechemically synthesized or are commercially available.

In certain embodiments, R^(1e) is hydrogen. In certain embodiments,R^(1c) is C₁₋₆alkyl.

In certain embodiments, R^(1c) is hydrogen. In certain embodiments,R^(1c) is alkyl. In certain embodiments, R^(1c) is aryl.

In Formula (I), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (I), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (I), R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂,and optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen. In certain embodiments, R⁴ is hydrogen. Incertain embodiments, R⁴ is —OH. In certain embodiments, R⁴ is NH₂ or N₃.In certain embodiments, R⁴ is —CH═CH₂. In certain embodiments, R⁴ isC₁₋₂ alkyl. In certain embodiments, R⁴ is substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH. In certain embodiments, R⁴ issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃. Incertain embodiments, R⁴ is substituted C₁₋₂ alkyl, wherein alkyl issubstituted with halogen.

In Formula (I), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments. R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (I), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷ ishydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (I), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

B. Formula II

The present disclosure provides a compound of Formula (II):

wherein

X¹ is NH or O;

X² is NH or O;

R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl, optionallysubstituted C₁₋₂₀alkenyl, or optionally substituted C₁₋₂₀alkynyl;

R¹ is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl;

R^(1c) is selected from hydrogen, alkyl, and aryl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃,or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X³ is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, R¹ carries a positive charge or a negativecharge. In certain embodiments, the compound of Formula (II) is azwitterion.

In certain embodiments, the compound of formula (II) is the enriched orisolated (R) enantiomer at the stereocenter bearing R⁴.

In Formula (II), X¹ is NH or O. In certain embodiments, X¹ is NH. Incertain embodiments, X¹ is O.

In Formula (II), X² is NH or O. In certain embodiments, X² is NH. Incertain embodiments, X² is O.

In Formula (II), R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl,optionally substituted C₁₋₂₀alkenyl, or optionally substitutedC₁₋₂₀alkynyl. In certain embodiments, R³⁰ is hydrogen. In certainembodiments, R³⁰ is C₁₋₂₀alkyl, C₁₋₂₀alkenyl, or C₁₋₂₀alkynyl. Incertain embodiments, R³⁰ is C₁₋₂₀alkyl. In certain embodiments, R³⁰ isC₁₋₁₅alkyl. In certain embodiments, R³⁰ is C₁₋₁₀alkyl. In certainembodiments, R³⁰ is C₁₋₂₀alkenyl. In certain embodiments, R³⁰ isC₁₋₁₅alkenyl. In certain embodiments, R³⁰ is C₁₋₁₀alkenyl. In certainembodiments, R³⁰ is C₁₋₂₀alkynyl. In certain embodiments, R³⁰ isC₁₋₁₅alkynyl. In certain embodiments, R³⁰ is C₁₋₁₀alkynyl.

In Formula (II), R¹ is selected from —NR^(1a)R^(1b) and OR^(1c). Incertain embodiments, R¹ is —NR^(1a)R^(1b). In certain embodiments, R¹ isOR^(1c). In certain embodiments, one of R^(1a) and R^(1b) is apolyamine.

In certain embodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₅₋₂₀alkyl, C₁₀₋₂₀alkyl,C₁₅₋₂₀alkyl, C₁₋₁₅alkyl, C₁₋₁₀alkyl, C₁₋₅alkyl, or C₅₋₁₅alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₈alkyl, C₉alkyl,C₁₀alkyl, C₁₁alkyl, or C₁₂alkyl.

In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or —(CH₂)NH₂;and n is independently a number from 2 to 4. In certain embodiments, oneof R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In certainembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NH₂, wherein n is anumber from 2 to 4.

In certain embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In certain instances, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl. In certain instances, R^(1d) is selectedfrom hydrogen, alkyl, substituted alkyl, heteroaryl, and substitutedheteroaryl. In certain instances, R^(1d) is alkyl or substituted alkyl.

In certain instances, R^(1d) is an amino acid side chain. In certaininstances, —CH(R^(1d))—C(O)OR^(1e) is an amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. In certain instances, R^(1d) is a positively charged amino acidside chain. In certain instances, —CH(R^(1d))—C(O)OR^(1e) is an aminoacid selected from lysine, arginine, and histidine.

Amino acid refers to the natural (genetically encoded) or unnatural orsynthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “natural” refers to thegenetically encoded 20 amino acids in their natural configuration.“Unnatural amino acids” have been modified after protein synthesis,and/or have a chemical structure in their side chain(s) different fromthat of the standard amino acids. Unnatural amino acids can bechemically synthesized or are commercially available.

In certain embodiments, R^(1e) is hydrogen. In certain embodiments,R^(1e) is C₁₋₆alkyl.

In certain embodiments, R^(1c) is hydrogen. In certain embodiments,R^(1c) is alkyl. In certain embodiments, R^(1c) is aryl.

In Formula (II), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (II), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (II), R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂,and optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen. In certain embodiments, R⁴ is hydrogen. Incertain embodiments, R⁴ is —OH. In certain embodiments, R⁴ is NH₂ or N₃.In certain embodiments, R⁴ is —CH═CH₂. In certain embodiments, R⁴ isC₁₋₂ alkyl. In certain embodiments, R⁴ is substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH. In certain embodiments, R⁴ issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃. Incertain embodiments, R⁴ is substituted C₁₋₂ alkyl, wherein alkyl issubstituted with halogen.

In Formula (II), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (II), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷ ishydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (II), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

C. Formula III

The present disclosure provides a compound of Formula (III):

wherein

R¹ and R² are independently selected from —NR^(1a)R^(1b) and OR^(1c);wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl;

R^(1c) is selected from hydrogen, alkyl, and aryl;

wherein at least one of R¹ and R² is —NR^(1a)R^(1b);

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(6a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, R¹ is different from R². In certain embodiments,one of R¹ and R² carries a positive charge and the other carries anegative charge. In certain embodiments, the compound of Formula (III)is a zwitterion.

In certain embodiments, if the compound of formula (III) bears astereocenter at R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then thecompound can be an enriched or isolated (R) enantiomer at a stereocenterbearing R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b). In certainembodiments, if the compound of formula (III) bears a stereocenter atR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then the compound can bean enriched or isolated (S) enantiomer at a stereocenter bearingR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b).

In Formula (III), R¹ and R² are independently selected from—NR^(1a)R^(1b) and OR^(1c); wherein at least one of R¹ and R² is—NR^(1a)R^(1b). In certain embodiments, R¹ is —NR^(1a)R^(1b) and R² isOR^(1c). In certain embodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl;and R² is OH. In certain embodiments, one of R^(1a) and R^(1b) is apolyamine; and R² is OH.

In certain embodiments, one of R^(1a) and R^(1b) is hydrogen. In certainembodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl. In certaininstances, one of R^(1a) and R^(1b) is C₅₋₂₀alkyl, C₁₋₂₀alkyl,C₁₅₋₂₀alkyl, C₁₋₁₅alkyl, C₁₋₁₀alkyl, C₁₋₅alkyl, or C₅₋₁₅alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₈alkyl, C₉alkyl,C₁₀alkyl, C₁₁alkyl, or C₁₂alkyl.

In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or —(CH₂)NH₂;and n is independently a number from 2 to 4. In certain embodiments, oneof R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In certainembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NH₂, wherein n is anumber from 2 to 4.

In certain embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In certain instances, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl. In certain instances, R^(1d) is selectedfrom hydrogen, alkyl, substituted alkyl, heteroaryl, and substitutedheteroaryl. In certain instances, R^(1d) is alkyl or substituted alkyl.

In certain instances, R^(1d) is an amino acid side chain. In certaininstances, —CH(R^(1d))—C(O)OR^(1e) is an amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. In certain instances, R^(1d) is a positively charged amino acidside chain. In certain instances, —CH(R^(1d))—C(O)OR^(1e) is an aminoacid selected from lysine, arginine, and histidine.

Amino acid refers to the natural (genetically encoded) or unnatural orsynthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “natural” refers to thegenetically encoded 20 amino acids in their natural configuration.“Unnatural amino acids” have been modified after protein synthesis,and/or have a chemical structure in their side chain(s) different fromthat of the standard amino acids. Unnatural amino acids can bechemically synthesized or are commercially available.

In certain embodiments, R^(1e) is hydrogen. In certain embodiments,R^(1e) is C₁₋₆alkyl.

In certain embodiments, R^(1c) is hydrogen. In certain embodiments,R^(1c) is alkyl. In certain embodiments, R^(1c) is aryl.

In Formula (III), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (III), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (III), R^(4a) and R^(4b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(4a) and R^(4b) are hydrogen. In certain embodiments, oneof R^(4a) and R^(4b) is —OH. In certain embodiments, one of R^(4a) andR^(4b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(4a) and R^(4b) is —CH═CH₂. In certain embodiments, one of R^(4a) andR^(4b) is C₁₋₂ alkyl. In certain embodiments, one of R^(4a) and R^(4b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(4a) and R^(4b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(4a) is hydrogen. In certain embodiments,R^(4a) is —OH. In certain embodiments, R^(4a) is NH₂ or N₃. In certainembodiments, R^(4a) is —CH═CH₂. In certain embodiments, R^(4a) is C₁₋₂alkyl. In certain embodiments, R^(4a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(4b) is hydrogen. In certain embodiments,R^(4b) is —OH. In certain embodiments, R^(4b) is NH₂ or N₃. In certainembodiments, R^(4b) is —CH═CH₂. In certain embodiments, R^(4b) is C₁₋₂alkyl. In certain embodiments, R^(4b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (III), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (III), R^(6a) and R^(6h) are independently selected fromhydrogen. —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH. —NH₂, N₃, or halogen. In certainembodiments, R^(6a) and R^(6b) are hydrogen. In certain embodiments, oneof R^(6a) and R^(6b) is —OH. In certain embodiments, one of R^(6a) andR^(6b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(6a) and R^(6b) is —CH═CH₂. In certain embodiments, one of R^(6a) andR^(6b) is C₁₋₂ alkyl. In certain embodiments, one of R^(6a) and R^(6b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(6a) and R^(6b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(6a) is hydrogen. In certain embodiments,R^(6a) is —OH. In certain embodiments, R^(6a) is NH₂ or N₃. In certainembodiments, R^(6a) is —CH═CH₂. In certain embodiments, R^(6a) is C₁₋₂alkyl. In certain embodiments, R^(6a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(6b) is hydrogen. In certain embodiments,R^(6b) is —OH. In certain embodiments, R^(6b) is NH₂ or N₃. In certainembodiments, R^(6b) is —CH═CH₂. In certain embodiments, R^(6b) is C₁₋₂alkyl. In certain embodiments, R^(6b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (III), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷is hydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (III), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

D. Formula IV

The present disclosure provides a compound of Formula (IV):

wherein

X¹ is NH or O;

X² is NH or O;

R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl, optionallysubstituted C₁₋₂₀alkenyl, or optionally substituted C₁₋₂₀alkynyl;

R¹ is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl;

R^(1c) is selected from hydrogen, alkyl, and aryl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(6a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X³ is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, R¹ carries a positive charge or a negativecharge. In certain embodiments, the compound of Formula (IV) is azwitterion.

In certain embodiments, if the compound of formula (IV) bears astereocenter at R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then thecompound can be an enriched or isolated (R) enantiomer at a stereocenterbearing R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b). In certainembodiments, if the compound of formula (IV) bears a stereocenter atR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then the compound can bean enriched or isolated (S) enantiomer at a stereocenter bearingR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b).

In Formula (IV), X¹ is NH or O. In certain embodiments, X¹ is NH. Incertain embodiments, X¹ is O.

In Formula (IV), X² is NH or O. In certain embodiments, X² is NH. Incertain embodiments, X² is O.

In Formula (IV), R³⁰ is hydrogen, optionally substituted C₁₋₂₀alkyl,optionally substituted C₁₋₂₀alkenyl, or optionally substitutedC₁₋₂₀alkynyl. In certain embodiments, R³⁰ is hydrogen. In certainembodiments, R³⁰ is C₁₋₂₀alkyl, C₁₋₂₀alkenyl, or C₁₋₂₀alkynyl. Incertain embodiments, R³⁰ is C₁₋₂₀alkyl. In certain embodiments, R³⁰ isC₁₋₁₅alkyl. In certain embodiments, R³⁰ is C₁₋₁₀alkyl. In certainembodiments, R³⁰ is C₁₋₂₀alkenyl. In certain embodiments, R³⁰ isC₁₋₁₅alkenyl. In certain embodiments, R³⁰ is C₁₋₁₀alkenyl. In certainembodiments, R³⁰ is C₁₋₂₀alkynyl. In certain embodiments, R³⁰ isC₁₋₁₅alkynyl. In certain embodiments, R³⁰ is C₁₋₁₀alkynyl.

In Formula (IV), R¹ is selected from —NR^(1a)R^(1b) and OR^(1c). Incertain embodiments, R¹ is —NR^(1a)R^(1b). In certain embodiments, R¹ isOR^(1c). In certain embodiments, one of R^(1a) and R^(1b) is apolyamine.

In certain embodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₅₋₂₀alkyl, C₁₁₋₂₀alkyl,C₁₅₋₂₀alkyl, C₁₋₁₅alkyl, C₁₋₁₀alkyl, C₁₋₅alkyl, or C₅₋₁₅alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₈alkyl, C₉alkyl,C₁₀alkyl, C₁₁alkyl, or C₁₂alkyl.

In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In certainembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); whereinR^(x) is hydrogen or —(CH₂)_(n)NH₂; and n is independently a number from2 to 4. In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH₂, wherein n is a number from 2 to 4.

In certain embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In certain instances, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl. In certain instances, R^(1d) is selectedfrom hydrogen, alkyl, substituted alkyl, heteroaryl, and substitutedheteroaryl. In certain instances, R^(1d) is alkyl or substituted alkyl.

In certain instances, R^(1d) is an amino acid side chain. In certaininstances, —CH(R^(1d))—C(O)OR^(1e) is an amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. In certain instances, R^(1d) is a positively charged amino acidside chain. In certain instances, —CH(R^(1d))—C(O)OR^(1e) is an aminoacid selected from lysine, arginine, and histidine.

Amino acid refers to the natural (genetically encoded) or unnatural orsynthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “natural” refers to thegenetically encoded 20 amino acids in their natural configuration.“Unnatural amino acids” have been modified after protein synthesis,and/or have a chemical structure in their side chain(s) different fromthat of the standard amino acids. Unnatural amino acids can bechemically synthesized or are commercially available.

In certain embodiments, R^(1e) is hydrogen. In certain embodiments,R^(1e) is C₁₋₆alkyl.

In certain embodiments, R^(1c) is hydrogen. In certain embodiments,R^(1c) is alkyl. In certain embodiments, R^(1c) is aryl.

In Formula (IV), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (IV), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (IV), R^(4a) and R^(4b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(4a) and R^(4b) are hydrogen. In certain embodiments, oneof R^(4a) and R^(4b) is —OH. In certain embodiments, one of R^(4a) andR^(4b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(4a) and R^(4b) is —CH═CH₂. In certain embodiments, one of R^(4a) andR^(4b) is C₁₋₂ alkyl. In certain embodiments, one of R^(4a) and R^(4b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(4a) and R^(4b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(4a) is hydrogen. In certain embodiments,R^(4a) is —OH. In certain embodiments, R^(4a) is NH₂ or N₃. In certainembodiments, R^(4a) is —CH═CH₂. In certain embodiments, R^(4a) is C₁₋₂alkyl. In certain embodiments, R^(4a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(4b) is hydrogen. In certain embodiments,R^(4b) is —OH. In certain embodiments, R^(4b) is NH₂ or N₃. In certainembodiments, R^(4b) is —CH═CH₂. In certain embodiments, R^(4b) is C₁₋₂alkyl. In certain embodiments, R^(4b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (IV), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁-2 alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (IV), R^(6a) and R^(6b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(6a) and R^(6b) are hydrogen. In certain embodiments, oneof R^(6a) and R^(6b) is —OH. In certain embodiments, one of R^(6a) andR^(6b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(6a) and R^(6b) is —CH═CH₂. In certain embodiments, one of R^(6a) andR^(6b) is C₁₋₂ alkyl. In certain embodiments, one of R^(6a) and R^(6b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(6a) and R^(6b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(6a) is hydrogen. In certain embodiments,R^(6a) is —OH. In certain embodiments, R^(6a) is NH₂ or N₃. In certainembodiments, R^(6a) is —CH═CH₂. In certain embodiments, R^(6a) is C₁₋₂alkyl. In certain embodiments, R^(6a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(6b) is hydrogen. In certain embodiments,R^(6b) is —OH. In certain embodiments, R^(6b) is NH₂ or N₃. In certainembodiments, R^(6b) is —CH═CH₂. In certain embodiments, R^(6b) is C₁₋₂alkyl. In certain embodiments, R^(6b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (IV), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷ ishydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (IV), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

E. Formula V

The present disclosure provides a compound of Formula (V) for use in themethods of the embodiments:

wherein

R¹ and R² are independently selected from —OH, monophosphate,diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) areindependently selected from hydrogen and C₁₋₅alkyl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R⁴ is selected from —OH, —NH₂, N₃, —CH═CH₂, and optionally substitutedC₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen. —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, the compound of formula (V) is the enriched orisolated (R) enantiomer at the stereocenter bearing R⁴.

In Formula (V), R¹ and R² are independently selected from —OH,monophosphate, diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a)and R^(1b) are independently selected from hydrogen and C₁₋₅alkyl. Incertain embodiments, R¹ and R² are —OH. In certain embodiments, one ofR¹ and R² is monophosphate. In certain embodiments, one of R¹ and R² isdiphosphate. In certain embodiments, one of R¹ and R² is—OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) are independentlyselected from hydrogen and C₁₋₅alkyl.

In certain embodiments, one of R¹ and R² is monophosphate and the otheris —OH. In certain embodiments, one of R¹ and R² is diphosphate and theother is —OH. In certain embodiments, one of R¹ and R² is—OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) are independentlyselected from hydrogen and C₁₋₅alkyl and the other is —OH.

In Formula (V), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (V), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (V), R⁴ is selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂,and optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen. In certain embodiments, R⁴ is hydrogen. Incertain embodiments, R⁴ is —OH. In certain embodiments, R⁴ is NH₂ or N₃.In certain embodiments, R⁴ is —CH═CH₂. In certain embodiments, R⁴ isC₁₋₂ alkyl. In certain embodiments, R⁴ is substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH. In certain embodiments, R⁴ issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃. Incertain embodiments, R⁴ is substituted C₁₋₂ alkyl, wherein alkyl issubstituted with halogen.

In Formula (V), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (V), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷ ishydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (V), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

F. Formula VI

The present disclosure provides a compound of Formula (VI) for use inthe methods of the embodiments:

wherein

R¹ and R² are independently selected from —OH, monophosphate,diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) areindependently selected from hydrogen and C₁₋₅alkyl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R^(6a) and R^(6b) are independently selected from hydrogen. —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂. N₃, or halogen:

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, if the compound of formula (VI) bears astereocenter at R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then thecompound can be an enriched or isolated (R) enantiomer at a stereocenterbearing R^(4a)/R^(4b). R^(5a)/R^(5b), or R^(6a)/R^(6b). In certainembodiments, if the compound of formula (VI) bears a stereocenter atR^(4a)/R^(4b). R^(5a)/R^(5b), or R^(6a)/R^(6b), then the compound can bean enriched or isolated (S) enantiomer at a stereocenter bearingR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b).

In Formula (VI), R¹ and R² are independently selected from —OH,monophosphate, diphosphate or —OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a)and R^(1b) are independently selected from hydrogen and C₁₋₅alkyl. Incertain embodiments, R¹ and R² are —OH. In certain embodiments, one ofR¹ and R² is monophosphate. In certain embodiments, one of R¹ and R² isdiphosphate. In certain embodiments, one of R¹ and R² is—OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) are independentlyselected from hydrogen and C₁₋₅alkyl.

In certain embodiments, one of R¹ and R² is monophosphate and the otheris —OH. In certain embodiments, one of R¹ and R² is diphosphate and theother is —OH. In certain embodiments, one of R¹ and R² is—OCH(R^(1b))OC(O)OR^(1a); wherein R^(1a) and R^(1b) are independentlyselected from hydrogen and C₁₋₅alkyl and the other is —OH.

In Formula (VI), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (VI), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (VI), R^(4a) and R^(4b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(4a) and R^(4b) are hydrogen. In certain embodiments, oneof R^(4a) and R^(4b) is —OH. In certain embodiments, one of R^(4a) andR^(4b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(4a) and R^(4b) is —CH═CH₂. In certain embodiments, one of R^(4a) andR^(4b) is C₁₋₂ alkyl. In certain embodiments, one of R^(4a) and R^(4b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(4a) and R^(4b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(4a) is hydrogen. In certain embodiments,R^(4a) is —OH. In certain embodiments, R^(4a) is NH₂ or N₃. In certainembodiments, R^(4a) is —CH═CH₂. In certain embodiments, R^(4a) is C₁₋₂alkyl. In certain embodiments, R^(4a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(4b) is hydrogen. In certain embodiments,R^(4b) is —OH. In certain embodiments, R^(4b) is NH₂ or N₃. In certainembodiments, R^(4b) is —CH═CH₂. In certain embodiments, R^(4b) is C₁₋₂alkyl. In certain embodiments, R^(4b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (VI), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (VI), R^(6a) and R^(6b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(6a) and R^(6b) are hydrogen. In certain embodiments, oneof R^(6a) and R^(6b) is —OH. In certain embodiments, one of R^(6a) andR^(6b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(6a) and R^(6b) is —CH═CH₂. In certain embodiments, one of R^(6a) andR^(6b) is C₁₋₂ alkyl. In certain embodiments, one of R^(6a) and R^(6b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(6a) and R^(6b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(6a) is hydrogen. In certain embodiments,R^(6a) is —OH. In certain embodiments, R^(6a) is NH₂ or N₃. In certainembodiments, R^(6a) is —CH═CH₂. In certain embodiments, R^(6a) is C₁₋₂alkyl. In certain embodiments, R^(6a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(6b) is hydrogen. In certain embodiments,R^(6b) is —OH. In certain embodiments, R^(6b) is NH₂ or N₃. In certainembodiments, R^(6b) is —CH═CH₂. In certain embodiments, R^(6b) is C₁₋₂alkyl. In certain embodiments, R^(6b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (VI), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷ ishydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (VI), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

G. Formula VII

The present disclosure provides a compound of Formula (VII):

wherein

R² is selected from —NR^(1a)R^(1b) and OR^(1c); wherein

R^(1a) and R^(1b) are independently selected from hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted polyamine, and—CH(R^(1d))—C(O)OR^(1e), wherein

-   -   R^(1d) is selected from hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        heteroalkyl, substituted heteroalkyl heteroaryl, substituted        heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl;        and    -   R^(1e) is hydrogen or C₁₋₆alkyl;

R^(1c) is selected from hydrogen, alkyl, and aryl;

R^(3a) and R^(3b) are independently selected from hydrogen and halo;

W is O, S, or NH;

R^(4a) and R^(4b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH. —NH₂, N₃, or halogen;

R^(5a) and R^(5b) are independently selected from hydrogen, —OH. —NH₂.N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen:

R^(6a) and R^(4b) are independently selected from hydrogen. —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen;

R⁷ is hydrogen or fluoro; and

X is O, S, or NH;

L is an optionally substituted polyamine;

m is zero or one;

n is zero or one;

and salts, hydrates, solvates, and tautomers, thereof.

In certain embodiments, if the compound of formula (VII) bears astereocenter at R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then thecompound can be an enriched or isolated (R) enantiomer at a stereocenterbearing R^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b) In certainembodiments, if the compound of formula (VII) bears a stereocenter atR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b), then the compound can bean enriched or isolated (S) enantiomer at a stereocenter bearingR^(4a)/R^(4b), R^(5a)/R^(5b), or R^(6a)/R^(6b).

In Formula (VII), R² is selected from —NR^(1a)R^(1b) and OR^(1c). Incertain embodiments, R² is —NR^(1a)R^(1b). In certain embodiments, R² isOR^(1c). In certain embodiments, one of R^(1a) and R^(1b) is apolyamine.

In certain embodiments, one of R^(1a) and R^(1b) is C₁₋₂₀alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₅₋₂₀alkyl, C₁₀₋₂₀alkyl,C₁₅₋₂₀alkyl, C₁₋₁₅alkyl, C₁₋₁₀alkyl, C₁₋₅alkyl, or C₅₋₁₅alkyl. Incertain instances, one of R^(1a) and R^(1b) is C₈alkyl, C₉alkyl,C₁₀alkyl, C₁₁alkyl, or C₁₂alkyl.

In certain embodiments, one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)NHR^(x); wherein R^(x) is hydrogen or —(CH₂)_(n)NH₂;and n is independently a number from 2 to 4. In certain embodiments, oneof R^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x); wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂; and n is independently a number from 2 to 4. In certainembodiments, one of R^(1a) and R^(1b) is —(CH₂)_(n)NH₂, wherein n is anumber from 2 to 4.

In certain embodiments, one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH. In certain instances, R^(1d) isselected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, andsubstituted heteroarylalkyl. In certain instances, R^(1d) is selectedfrom hydrogen, alkyl, substituted alkyl, heteroaryl, and substitutedheteroaryl. In certain instances, R^(1d) is alkyl or substituted alkyl.

In certain instances, R^(1d) is an amino acid side chain. In certaininstances, —CH(R^(1d))—C(O)OR^(1e) is an amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. In certain instances, R^(1d) is a positively charged amino acidside chain. In certain instances, —CH(R^(1d))—C(O)OR^(1e) is an aminoacid selected from lysine, arginine, and histidine.

Amino acid refers to the natural (genetically encoded) or unnatural orsynthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “natural” refers to thegenetically encoded 20 amino acids in their natural configuration.“Unnatural amino acids” have been modified after protein synthesis,and/or have a chemical structure in their side chain(s) different fromthat of the standard amino acids. Unnatural amino acids can bechemically synthesized or are commercially available.

In certain embodiments, R^(1e) is hydrogen. In certain embodiments,R^(1e) is C₁₋₆alkyl.

In certain embodiments, R^(1c) is hydrogen. In certain embodiments,R^(1c) is alkyl. In certain embodiments, R^(1c) is aryl.

In Formula (VII), R^(3a) and R^(3b) are independently selected fromhydrogen and halo. In certain embodiments, R^(3a) and R^(3b) arehydrogen. In certain embodiments, one of R^(3a) and R^(3b) is halo.

In Formula (VII), W is O, S, or NH. In certain embodiments, W is O. Incertain embodiments, W is S. In certain embodiments, W is NH.

In Formula (VII), R^(4a) and R^(4b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(4a) and R^(4b) are hydrogen. In certain embodiments, oneof R^(4a) and R^(4b) is —OH. In certain embodiments, one of R^(4a) andR^(4b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(4a) and R^(4b) is —CH═CH₂. In certain embodiments, one of R^(4a) andR^(4b) is C₁₋₂ alkyl. In certain embodiments, one of R^(4a) and R^(4b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(4a) and R^(4b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(4a) is hydrogen. In certain embodiments,R^(4a) is —OH. In certain embodiments, R^(4a) is NH₂ or N₃. In certainembodiments, R^(4a) is —CH═CH₂. In certain embodiments, R^(4a) is C₁₋₂alkyl. In certain embodiments, R^(4a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(4b) is hydrogen. In certain embodiments,R^(4b) is —OH. In certain embodiments, R^(4b) is NH₂ or N₃. In certainembodiments, R^(4b) is —CH═CH₂. In certain embodiments, R^(4b) is C₁₋₂alkyl. In certain embodiments, R^(4b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(4b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (VII), R^(5a) and R^(5b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(5a) and R^(5b) are hydrogen. In certain embodiments, oneof R^(5a) and R^(5b) is —OH. In certain embodiments, one of R^(5a) andR^(5b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(5a) and R^(5b) is —CH═CH₂. In certain embodiments, one of R^(5a) andR^(5b) is C₁₋₂ alkyl. In certain embodiments, one of R^(5a) and R^(5b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(5a) and R^(5b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(5a) is hydrogen. In certain embodiments,R^(5a) is —OH. In certain embodiments, R^(5a) is NH₂ or N₃. In certainembodiments, R^(5a) is —CH═CH₂. In certain embodiments, R^(5a) is C₁₋₂alkyl. In certain embodiments, R^(5a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5a) issubstituted C₁₋₂alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(5b) is hydrogen. In certain embodiments,R^(5b) is —OH. In certain embodiments, R^(5b) is NH₂ or N₃. In certainembodiments, R^(5b) is —CH═CH₂. In certain embodiments, R^(5b) is C₁₋₂alkyl. In certain embodiments, R^(5b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(5b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In Formula (VII), R^(6a) and R^(6b) are independently selected fromhydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl,wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen. In certainembodiments, R^(6a) and R^(6b) are hydrogen. In certain embodiments, oneof R^(6a) and R^(6b) is —OH. In certain embodiments, one of R^(6a) andR^(6b) is selected from —NH₂, and N₃. In certain embodiments, one ofR^(6a) and R^(6b) is —CH═CH₂. In certain embodiments, one of R^(6a) andR^(6b) is C₁₋₂ alkyl. In certain embodiments, one of R^(6a) and R^(6b)is optionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, or N₃. In certain embodiments, one of R^(6a) and R^(6b) isoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted withhalogen.

In certain embodiments, R^(6a) is hydrogen. In certain embodiments,R^(6a) is —OH. In certain embodiments, R^(6a) is NH₂ or N₃. In certainembodiments, R^(6a) is —CH═CH₂. In certain embodiments, R^(6a) is C₁₋₂alkyl. In certain embodiments, R^(6a) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6a) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, R^(6b) is hydrogen. In certain embodiments,R^(6b) is —OH. In certain embodiments, R^(6b) is NH₂ or N₃. In certainembodiments, R^(6b) is —CH═CH₂. In certain embodiments, R^(6b) is C₁₋₂alkyl. In certain embodiments, R^(6b) is substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH. In certain embodiments, R^(6b) issubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —NH₂ or N₃.

In certain embodiments, m is zero. In certain embodiments, m is one. Incertain embodiments, n is zero. In certain embodiments, n is one.

In Formula (VII), R⁷ is hydrogen or fluoro. In certain embodiments, R⁷is hydrogen. In certain embodiments, R⁷ is fluoro.

In Formula (VII), X is O, S, or NH. In certain embodiments, X is O. Incertain embodiments, X is S. In certain embodiments, X is NH.

In Formula (VII), L¹ is an optionally substituted polyamine. In certainembodiments, L¹ is a polyamine. In certain embodiments, L¹ is—NH—(CH₂)_(n)—NH—(CH₂)_(n)—NH—(CH₂)_(n)—NH—; and n is independently anumber from 2 to 4. In certain embodiments, L¹ is—NH—(CH₂)_(n)—NH—(CH₂)_(n)—NH—; and n is independently a number from 2to 4. In certain embodiments, L¹ is —NH—(CH₂)_(n)—NH—; and n isindependently a number from 2 to 4.

The present disclosure provides compounds as shown in Tables 9 and 10and their use in the methods of the embodiments.

The present disclosure provides compounds of following formulae andtheir use in the methods of the embodiments.

The present disclosure provides compounds of following formulae andtheir use in the methods of the embodiments.

The present disclosure provides compounds of following formulae andtheir use in the methods of the embodiments.

The present disclosure provides compounds of following formulae andtheir use in the methods of the embodiments.

The present disclosure provides compounds of following formulae andtheir use in the methods of the embodiments.

The compounds of Formulae (I)-(IX) may be prepared and/or formulated aspharmaceutically acceptable salts. Pharmaceutically acceptable salts arenon-toxic salts of a free base form of a compound that possesses thedesired pharmacological activity of the free base. These salts may bederived from inorganic or organic acids. Non-limiting examples ofpharmaceutically acceptable salts include sulfates, pyrosulfates,bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides,bromides, iodides, acetates, propionates, decanoates, caprylates,acrylates, formates, isobutyrates, caproates, heptanoates, propiolates,oxalates, malonates, succinates, suberates, sebacates, fumarates,maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates,chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates,methoxybenzoates, phthalates, sulfonates, methylsulfonates,propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, and mandelates. Lists of other suitable pharmaceuticallyacceptable salts are found in Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Company, Easton, Pa., 1985.

For a compound of any one of Formulae (I)-(IX) that contains a basicnitrogen, a pharmaceutically acceptable salt may be prepared by anysuitable method available in the art, for example, treatment of the freebase with an inorganic acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoricacid, and the like, or with an organic acid, such as acetic acid,phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbicacid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid,valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid,glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha-hydroxy acid, such as mandelic acid, citric acid, or tartaricacid, an amino acid, such as aspartic acid or glutamic acid, an aromaticacid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, orcinnamic acid, a sulfonic acid, such as laurylsulfonic acid,p-toluenesulfonic acid, methanesulfonic acid, or ethanesulfonic acid, orany compatible mixture of acids such as those given as examples herein,and any other acid and mixture thereof that are regarded as equivalentsor acceptable substitutes in light of the ordinary level of skill inthis technology.

G. Synthesis of Compounds

The embodiments are also directed to processes and intermediates usefulfor preparing subject compounds or a salt or solvate or stereoisomerthereof.

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001.)

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as high performanceliquid chromatography (HPLC), preparative thin layer chromatography,flash column chromatography and ion exchange chromatography. Anysuitable stationary phase can be used, including normal and reversedphases as well as ionic resins. Most typically the disclosed compoundsare purified via silica gel and/or alumina chromatography. See, e.g.,Introduction to Modern Liquid Chromatography, 2nd ed., ed. L. R. Snyderand J. J. Kirkland, John Wiley and Sons, 1979; and Thin LayerChromatography, E. Stahl (ed.), Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 4^(th) ed., Wiley, New York 2006. The protecting groups maybe removed at a convenient subsequent stage using methods known from theart.

Exemplary chemical entities useful in methods of the embodiments willnow be described by reference to illustrative synthetic schemes fortheir general preparation herein and the specific examples that follow.Artisans will recognize that, to obtain the various compounds herein,starting materials may be suitably selected so that the ultimatelydesired substituents will be carried through the reaction scheme with orwithout protection as appropriate to yield the desired product.Alternatively, it may be necessary or desirable to employ, in the placeof the ultimately desired substituent, a suitable group that may becarried through the reaction scheme and replaced as appropriate with thedesired substituent. Furthermore, one of skill in the art will recognizethat the transformations shown in the schemes below may be performed inany order that is compatible with the functionality of the particularpendant groups. Each of the reactions depicted in the general schemes ispreferably run at a temperature from about 0° C. to the refluxtemperature of the organic solvent used. Unless otherwise specified, thevariables are as defined above in reference to Formula (I).

Representative syntheses of compounds of the present disclosure aredescribed in schemes below, and the particular examples that follow.

Scheme 1 shows a representative synthesis of the compounds of theembodiments.

In Scheme 1, Compound 1-A is commercially available or can besynthesized by one of skill in the art. In Compound 1-A, Y is a leavinggroup. In certain instances, Y is a halogen, such as chloro. Compound1-A reacts with R-(+)-propylene carbonate in the presence of base toform Compound 1-B. In Scheme 1, propylene carbonate is shown withR-(+)-stereochemistry. However, the stereochemistry is shown forrepresentative purposes. Other stereochemistry or a racemic mixture forpropylene carbonate can also be used in the reaction scheme.

With continued reference to Scheme 1. Compound 1-B reacts with aphosphonylating agent to form Compound 1-C. The phosphonylating reactioncan be run in the presence of a strong base, such as lithiumtert-butoxide. In Compound 1-C, R^(y) is an alkyl group, such as methyl,ethyl, or propyl. Then, the alkyl groups on the phosphonate group areremoved to produce Compound 1-D. Conditions to remove the alkyl groupinclude standard reactions to remove alkyl protecting groups onphosphonates. Suitable conditions include reaction with acid hydrolysis,TMSBr, LiBr, or ammonium hydroxide.

With continued reference to Scheme 1, the leaving group Y of Compound1-D is converted to a heteroatom to yield Compound 1-E. Suitableconditions to convert Compound 1-D to Compound 1-E include hydrolysis,such as heating in an acidic solution.

Then, the phosphate group of Compound 1-E is alkylated to form Compound1-F. In Scheme 1, for representative purposes, the alkylation reagentadds the moiety-CH₂—O—C(O)—OR^(z) onto Compound 1-E. In certainembodiments, R^(z) is an alkyl group, such as methyl, ethyl, propyl, orisopropyl. Suitable conditions for alkylation of Compound 1-E includereaction in basic conditions with use of bases, such as triethylamineand TBAF.

An example of an experimental procedure to synthesize a compound of theembodiments is as follows. One millimole of a phosphonate compoundcomprising a moiety —O—CH₂—P(O)(OH)₂ was dried with coevaporation wassuspended in 30 mL NMP. One equivalent tetrabutylammonium bromide, 4equivalents of triethylamine, and 5 equivalents of chloromethylisopropyl carbonate (Santa Cruz Biotechnology, Santa Cruz. Calif.) wasadded and the reaction mixture heated for 5 hours at 50° C. Aftercooling, methylene chloride was added and the solution washed withbrine. After drying on sodium sulfate, the organic phase was evaporatedto about 30 ml. The NMP was removed by trituration with cyclohexane andhexane. The solid residue was purified by silica gel flashchromatography in methylene chloride containing 15 percent methanol.

The product can also be purified using HPLC C18 column, 50 mM TEAacetate, acetonitrile, the gradient 20-90 percent acetonitrile.

Scheme 2 shows a representative synthesis of the compounds of theembodiments.

In Scheme 2, Compound 2-A can be synthesized as shown in Scheme 1.Compound 2-A is reacted with carbonyldiimidazole to form Compound 2-Bcomprising an imidazole moiety. With an imidazole moiety as a leavinggroup, Compound 2-B can react with amino compounds to form Compound 2-C.For representative purposes, Scheme 2 shows reaction with spermine orspermidine to form Compound 2-C. However, any suitable amino compoundcan be used in Scheme 2.

Scheme 3 shows a representative synthesis of the compounds of theembodiments.

Scheme 3 shows a synthesis of an acyclic nucleoside phosphonatecompound. In Scheme 3, R is an alkyl group; X¹, X², and X³ are leavinggroups. Examples of leaving groups include halide, arylsulfonate,alkylsulfonate, and perfluoroalkylsulfonate.

Referring to Scheme 3. Compound 3-A is reacted with paraformaldehyde toform Compound 3-B. Reaction can occur in the presence of a base, such astriethylamine or diisopropylethylamine. Compound 3-B reacts with amethylpropene derivative with two leaving groups. In a first reaction,one of the leaving groups is displaced by the hydroxyl group of Compound3-B to from Compound 3-C. In a second reaction, the leaving group onCompound 3-C reacts with the amino group of a purine derivative to formCompound 3-D. Then, the leaving group of Compound 3-D is converted to aheteroatom to yield Compound 3-E. Suitable conditions to convertCompound 3-D to Compound 3-E include hydrolysis, such as heating in anacidic solution, when the leaving group is a halogen. Then, the alkylgroups of Compound 3-E are removed to form Compound 3-F. Suitableconditions to remove alkyl group from a phosphonate include use ofTMS-Br.

Scheme 4 shows a representative synthesis of the compounds of theembodiments.

In Scheme 4, —X—CH₂— is representative of —W—CH(R⁴)—C(R^(5a))(CR^(5b))—or —W—CH(R⁴)—C(R^(5b))—(CR^(5b))—C(R^(6a))(CR^(6b))— in the formulaeherein.

Referring to Scheme 4, Method A shows a synthesis of an acyclicnucleoside phosphonate polyamine compound using an imidazolyl derivativeas an intermediate. In Method A, Compound 4-A, an acyclic nucleosidephosphonate free acid, is reacted with carbonyldiimidazole to formCompound 4-B. Conditions for reaction with carbonyldiimidazole includereaction in an appropriate solvent in ambient temperature. Compound 4-Bis then reacted with a polyamine to produce Compound 4-D. The aminogroup of the polyamine can displace the imidazole moiety. Conditions forthe reaction with polyamine include reaction in an appropriate solventin ambient temperature.

With continued reference to Scheme 4, Method B shows a synthesis of anacyclic nucleoside phosphonate polyamine compound using a pyridinaminiumderivative as an intermediate. In Method B, Compound 4-A, an acyclicnucleoside phosphonate free acid, is reacted with 4-dimethylaminopyridin(DMAP) to form Compound 4-C. Conditions for reaction with DMAP includereaction with a base in an appropriate solvent in ambient to heatedtemperatures. An example of a suitable base is triphenylphosphine.Compound 4-C is then reacted with a polyamine to produce Compound 4-D.The amino group of the polyamine can displace the pyridinaminium moiety.Conditions for the reaction with polyamine include reaction in anappropriate solvent in ambient temperature.

Scheme 5 shows a representative synthesis of the compounds of theembodiments.

In Scheme 5, —X—CH₂— is representative of —W—CH(R⁴)—C(R^(5a))(CR^(5b))—or —W—CH(R⁴)—C(R^(5a))(CR^(5b))—C(R^(6a))(CR^(6b))— in the formulaeherein. In Scheme 5, Y is representative of a portion of the substituenton the phosphonate group of the compounds herein.

Referring to Scheme 5. Method C shows synthesis of both an acyclicnucleoside phosphonate polyamine compound and an acyclic bis-nucleosidephosphonate polyamine compound. In Method C, with use of a couplingreagent, both compounds can form. In Scheme 5. Compound 5-A reacts witha polyamine in the presence of a coupling reagent to form Compounds 5-Band 5-C. Suitable coupling reagents includebenzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluoro-phosphate(BOP),N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxibenzotriazolanhydrous (HOBt), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC)and the like.

H. Isomers, Salts, Solvates, Protected Forms, and Prodrugs

A certain compound may exist in one or more particular geometric,optical, enantiomeric, diastereomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (−) forms;keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- andanticlinal-forms; .alpha.- and .beta.-forms; axial and equatorial forms;boat-, chair-, twist-, envelope-, and halfchair-forms; and combinationsthereof, hereinafter collectively referred to as “isomers” (or “isomericforms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C.sub.1-7alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec- and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including racemic and other mixturesthereof. Methods for the preparation (e.g., asymmetric synthesis) andseparation (e.g., fractional crystallisation and chromatographic means)of such isomeric forms are either known in the art or are readilyobtained by adapting the methods taught herein in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate (e.g., hydrate), protected forms, andprodrugs thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 119.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: acetic, propionic, succinic, gycolic, stearic, palmitic,lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic,pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic,oxalic, isethionic, valeric, and gluconic. Examples of suitablepolymeric anions include, but are not limited to, those derived from thefollowing polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.,active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form,” as used herein, pertains to a compound in which one ormore reactive functional groups are protected from undesirable chemicalreactions, that is, are in the form of a protected or protecting group(also known as a masked or masking group). By protecting a reactivefunctional group, reactions involving other unprotected reactivefunctional groups can be performed, without affecting the protectedgroup; the protecting group may be removed, usually in a subsequentstep, without substantially affecting the remainder of the molecule.See, for example, Protective Groups in Organic Synthesis (T. Green andP. Wuts, Wiley, 1991), and Protective Groups in Organic Synthesis (T.Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃, —OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (>C═O) is convertedto a diether (>C(OR)₂), by reaction with, for example, a primaryalcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide(—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide(—NHCO—CH₃); a benzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxyamide (—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2-(phenylsulfonyl)ethyloxy amide (—NH-Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O.).

For example, a carboxylic acid group may be protected as an ester forexample, as: an C₁₋₇alkyl ester (e.g., a methyl ester; a t-butyl ester);a C₁₋₇haloalkyl ester (e.g., a C₁₋₇trihaloalkyl ester); atriC₁₋₇alkylsilyl-C₁₋₇alkyl ester; or a C₅₋₂₀aryl-C₁₋₇alkyl ester (e.g.,a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

I. Pharmaceutical Compositions

The present invention provides compounds that can specifically andpotently inhibit telomere elongation, and which may therefore be used toinhibit the proliferation of telomerase-positive cells, such as tumorcells. A very wide variety of cancer cells have been shown to betelomerase-positive, including cells from cancer of the skin, connectivetissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus,kidney, bladder, colon, prostate, central nervous system (CNS), retinaand hematologic tumors (such as myeloma, leukemia and lymphoma).

Accordingly, the compounds provided herein are broadly useful intreating a wide range of malignancies. More importantly, the compoundsof the present invention can be effective in providing treatments thatdiscriminate between malignant and normal cells to a high degree,avoiding many of the deleterious side-effects present with most currentchemotherapeutic regimens which rely on agents that kill dividing cellsindiscriminately. One aspect of the invention therefore is a method oftreating cancer in a patient, comprising administering to the patient atherapeutically effective dose of a compound of the present invention.Compounds of the invention, may be employed in conjunction with othercancer treatment approaches, including surgical removal of primarytumors, chemotherapeutic agents and radiation treatment

For therapeutic application, a compound of the invention is formulatedin a therapeutically effective amount with a pharmaceutically acceptablecarrier. One or more invention compounds may be included in any givenformulation. The pharmaceutical carrier may be solid or liquid. Liquidcarriers can be used in the preparation of solutions, emulsions,suspensions and pressurized compositions. The compounds are dissolved orsuspended or diluted in a pharmaceutically acceptable solid, semi-solid,or liquid excipient, which acts as a vehicle, carrier or medium for theactive ingredient. Suitable examples of liquid carriers for parenteraladministration of the compounds include water (which may containadditives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), phosphate buffered saline solution (PBS), alcohols(including monohydric alcohols and polyhydric alcohols, e.g., glycols)and their derivatives, and oils (e.g., fractionated coconut oil andarachis oil). The liquid carrier can contain other suitablepharmaceutical additives including, but not limited to, the following:solubilizers, suspending agents, emulsifiers, buffers, thickeningagents, colors, viscosity regulators, preservatives, stabilizers andosmolarity regulators.

For parenteral administration of the compounds, the carrier can also bean oily ester such as ethyl oleate and isopropyl myristate. Sterilecarriers are useful in sterile liquid form compositions for parenteraladministration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. The compounds can also beadministered intravascularly or via a vascular stent.

The liquid carrier for pressurized compositions can be a halogenatedhydrocarbon or other pharmaceutically acceptable propellant. Suchpressurized compositions may also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, the compounds may be formulated into anaqueous or partially aqueous solution, which can then be utilized in theform of an aerosol.

The compositions of the present invention can also be in the form oftablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders. Thecompounds may be administered topically as a solution, cream, or lotion,by formulation with pharmaceutically acceptable vehicles containing theactive compound. Some examples of suitable excipients or carriersinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,sterile water, syrup, and methyl cellulose. The formulations canadditionally include: lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying and suspendingagents; preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures known in the art.

In preparing a formulation, it may be necessary to mill the activelyophilized compound to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the active compound is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g. about 40mesh.

These pharmaceutical compositions of this invention can be administeredby a variety of routes including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular, and intranasal. Thesecompounds are effective as both injectable and oral compositions. Suchcompositions are prepared in a manner well known in the pharmaceuticalart and comprise at least one active compound. In some embodiments, thepharmaceutical compositions of this invention may be orally administeredin any acceptable dosage including, but not limited to, formulations incapsules, tablets, powders or granules, and as suspensions or solutionsin water or non-aqueous media. Pharmaceutical compositions and/orformulations comprising the compounds may include carriers, lubricants,diluents, thickeners, flavoring agents, emulsifiers, dispersing aids orbinders. In the case of tablets for oral use, carriers which arecommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For oral administrationin a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

When employed as oral compositions, the compounds of the presentinvention (such as any of the compounds useful for inhibiting telomereelongation described herein) can be protected from acid digestion in thestomach by a pharmaceutically acceptable protectant. For example, thetablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permit the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions cancontain suitable pharmaceutically acceptable excipients as describedsupra. The compositions can be administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents can be nebulized by use of inertgases. Nebulized solutions can be inhaled directly from the nebulizingdevice or the nebulizing device can be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions can also be administered, orally or nasally, fromdevices which deliver the formulation in an appropriate manner.

While the compounds of the invention have superior characteristics forcellular and tissue penetration, they may be formulated to provide evengreater benefit, for example in liposome carriers. The use of liposomesto facilitate cellular uptake is described, for example, in U.S. Pat.No. 4,897,355 and U.S. Pat. No. 4,394,448. Numerous publicationsdescribe the formulation and preparation of liposomes. The compounds canalso be formulated by mixing with additional penetration enhancers, suchas unconjugated forms of the lipid moieties described above, includingfatty acids and their derivatives. Examples include oleic acid, lauricacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein(a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonicacid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,acylcarnitines, acylcholines, mono- and di-glycerides andphysiologically acceptable salts thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.).

Complex formulations comprising one or more penetration enhancing agentsmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations. Exemplary combinations includechenodeoxycholic acid (CDCA), generally used at concentrations of about0.5 to 2%, combined with sodium caprate or sodium laurate, generallyused at concentrations of about 0.5 to 5%.

Pharmaceutical compositions and/or formulations comprising the compoundsof the present invention may also include chelating agents, surfactantsand non-surfactants. Chelating agents include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines). Surfactants include, for example, sodiumlauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether; and perfluorochemical emulsions, such asFC-43. Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives, and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone.

Thus, in another aspect of the invention, there is provided a method offormulating a pharmaceutical composition, the method comprisingproviding a compound as described herein, and combining the compoundwith a pharmaceutically acceptable excipient. Preferably the compound isprovided at pharmaceutical purity, as defined below. The method mayfurther comprise adding to the compound, either before or after theaddition of the excipient, a penetration enhancing agent.

The pharmaceutical composition will typically comply with pharmaceuticalpurity standards. For use as an active ingredient in a pharmaceuticalpreparation, a compound of this invention is generally purified awayfrom other reactive or potentially immunogenic components present in themixture in which they are prepared. Typically, to achieve pharmaceuticalpurity where a nucleic acid-based compound is the active ingredient, theactive ingredient is provided in at least about 50% homogeneity, andmore preferably 60%, 70%, 80% or 90% homogeneity, as determined byfunctional assay, chromatography, or gel electrophoresis. The activeingredient is then compounded into a medicament in accordance withgenerally accepted procedures for the preparation of pharmaceuticalpreparations. Thus, in the present invention, providing the compounds atpharmaceutical purity requires that the compound be provided at at leastabout 50% homogeneity, and more preferably at least 80% or 90%homogeneity.

The pharmaceutical composition will also typically be aliquoted andpackaged in either single dose or multi-dose units. The dosagerequirements for treatment with the compound vary with the particularcompositions employed, the route of administration, the severity of thesymptoms presented, the form of the compound and the particular subjectbeing treated.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 mg to about 100 mg or more, such as any of about1 mg to about 5 mg, 1 mg to about 10 mg, about 1 mg to about 20 mg,about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg toabout 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about1 mg to about 80 mg, or about 1 mg to about 90 mg, inclusive, includingany range in between these values, of the active ingredient. The term“unit dosage forms” refers to physically discrete units suitable asunitary dosages for individuals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient or carrier.

Pharmaceutical compositions of the invention can be administered to asubject in a formulation and in an amount effective to achieve aclinically desirable result. For the treatment of cancer, desirableresults include reduction in tumor mass (as determined by palpation orimaging; e.g., by radiography, radionucleotide scan, CAT scan, or MRI),reduction in the rate of tumor growth, reduction in the rate ofmetastasis formation (as determined e.g., by histochemical analysis ofbiopsy specimens), reduction in biochemical markers (including generalmarkers such as ESR, and tumor-specific markers such as serum PSA), andimprovement in quality of life (as determined by clinical assessment,e.g., Karnofsky score), increased time to progression, disease-freesurvival and overall survival.

The amount of compound per dose and the number of doses required toachieve such effects will vary depending on many factors including thedisease indication, characteristics of the patient being treated and themode of administration. Typically, the formulation and route ofadministration will provide a local concentration at the disease site ofbetween 1 μM and 1 nM of the compound. In general, the compounds areadministered at a concentration that affords effective results withoutcausing any harmful or deleterious side effects. Such a concentrationcan be achieved by administration of either a single unit dose, or bythe administration of the dose divided into convenient subunits atsuitable intervals throughout the day. The compounds of the presentinvention are effective over a wide dosage range and are generallyadministered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compounds actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

IV. Methods

Any of the compounds useful for inhibiting telomere elongation (such asin pharmaceutical compositions) provided herein are useful formodulating disease states associated with dysregulation of telomerelength. In some embodiments, the disease states associated withdysregulation of telomere length is a cell proliferative disorder isassociated with increased expression or activity of telomerase orcellular growth. In other embodiments, the cell proliferative disorderis cancer.

A. Methods for Inhibiting Telomere Elongation and Telomere ElongationInhibition Assays

The conjugates of the present invention may be used to inhibit or reducetelomere elongation and/or proliferation of cells having telomeraseactivity. In these contexts, inhibition or reduction of telomereextension or cell proliferation refer to a lower level of the measuredlength or activity relative to a control experiment in which the enzymeor cells are not treated with the conjugate. In particular embodiments,the inhibition or reduction in the measured length or activity is atleast a 10% reduction or inhibition. One of skill in the art willappreciate that reduction or inhibition of the measured length oractivity of at least 20%, 50%, 75%, 90% or 100% may be preferred forparticular applications. The ability of the invention compounds toinhibit telomere elongation can be determined in a cell-free assay(referred to as a biochemical assay) and in cells.

Accordingly, provided herein are methods for inhibiting telomereelongation in a cell. In one embodiment, the method comprises contactingthe cell with any of the compounds useful for inhibiting telomereelongation (including any of the pharmaceutical compositions) describedherein. In some embodiments, the cell is a cancer cell.

Also provided herein are methods for shortening telomere length in acell. In one embodiment, the method comprises contacting the cell withany of the compounds useful for inhibiting telomere elongation(including any of the pharmaceutical compositions) described herein. Insome embodiments, the cell is a cancer cell.

Methods for measuring inhibition of telomere elongation and the use ofsuch methods to determine the inhibitory activity of compounds aredescribed herein. For example, the TRAP assay is a standard assay methodfor measuring telomerase activity in a cell extract system and has beenwidely used in the search for telomerase inhibiting compounds (Kim etal., Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498,1997). The TRAP assay measures the amount of radioactive nucleotidesincorporated into elongation products (polynucleotides) formed bynucleotide addition to a telomerase substrate or primer. Theradioactivity incorporated can be measured as the intensity of a band ona detection screen (e.g., a Phosphorimager screen) exposed to a gel onwhich the radioactive products are separated. The TRAP assay is alsodescribed in detail in U.S. Pat. Nos. 5,629,154, 5,837,453 and5,863,726, and its use in testing the activity of telomerase inhibitorycompounds is described in various publications including WO 01/18015. Inaddition, the following kits are available commercially for researchpurposes for measuring telomerase activity: TRAPeze® XK TelomeraseDetection Kit (Cat. s7707; Intergen Co., Purchase N.Y.); and TeloTAGGGTelomerase PCR ELISA plus (Cat. 2,013,89; Roche Diagnostics,Indianapolis Ind.). The TRAP assay can be used to measure the inhibitionof telomere elongation rather than the inhibition of telomeraseactivity. If the present compounds are incorporated in the elongationproducts formed by nucleotide addition to a telomerase substrate orprimer, they will stop or “cap” the elongation product so thatadditional nucleotides cannot be added to the elongation product. Inthis way the elongation product is not extended beyond the compound andthe elongation products are only very short bands on a gel, rather thana ladder of different lengths of elongation products.

Another protocol for measuring the ability of compounds to inhibittelomere elongation in a biochemical assay is the direct (non-PCR based)cell-free telomerase assay, referred to as the “Flashplate assay”, anddescribed in Asai et al., Cancer Research, 63:3931-3939 (2003).

The ability of compounds of the invention to inhibit telomere elongationin cells may be determined by incubating the compound withtelomerase-expressing cells for a defined period of time, and thendetermining the telomere length in the cells. Telomerase-expressingtumor cell lines that are suitable for such assays include HME50-5Ehuman breast epithelial cells (provided by Dr. Jerry Shay, University ofTexas Southwestern Medical Center), the ovarian tumor cell lines OVCAR-5(MIISB, Milan) and SK-OV-3 (American Type Culture Collection, ATCC),human kidney carcinoma Caki-1 cells (Japanese Collection of ResearchBioresources, JCRB), human lung carcinoma 1549 cells (ATCC), humanepidermoid carcinoma A431 cells (JCRB), and human prostate cancer DU145cells (ATCC).

B. Cell Proliferation Assays

A key therapeutic application of the compounds of the invention is theinhibition of the growth of telomerase-expressing cells, particularlytumor cells. Compounds of the invention that inhibit telomere elongationin cells will induce crisis in telomerase-positive cell lines, leadingto cessation of cell growth and death. Importantly however, in normalhuman cells which do not express telomerase, such as BJ cells offibroblast origin, no crisis or other toxicity is induced by treatmentwith the invention compounds. The ability of the compounds tospecifically inhibit the growth of tumor cells can be assayed usingtumor cell lines in vitro, or in xenograft animal models in vivo.

A preferred protocol for such growth curve assays is the short term cellviability assay described in Asai et al. (2003). In selecting a compoundof the invention for therapeutic applications, it is preferred that thecompound produce no significant cytotoxic effects at concentrationsbelow about 10 μM in normal cells that do not express telomerase.

The ability of compounds of the invention to inhibit tumor cell growthin vivo can be confirmed using established xenograft models of humantumors, in which the test compound is administered either directly tothe tumor site or systemically, and the growth of the tumor is followedby physical measurement. Animals treated with compounds of the inventionare expected to have tumor masses that, on average, may increase for aperiod following the initial dosing, but will begin to shrink in masswith continuing treatment. In contrast, untreated control mice areexpected to have tumor masses that continue to increase. A preferredexample of a suitable in vivo tumor xenograft assay is described in Asaiet al. (2003). Other examples are described in Scorski et al., Proc.Natl. Acad. Sci. USA, 94: 3966-3971 (1997) and Damm et al., EMBO J.,20:6958-6968 (2001).

C. Cell Proliferative Disorders

A “proliferative disorder” is any cellular disorder in which the cellsproliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. The proliferative disorder includes, but is not limitedto, neoplasms. A “neoplasm” is an abnormal tissue growth, generallyforming a distinct mass that grows by cellular proliferation morerapidly than normal tissue growth. Neoplasms show partial or total lackof structural organization and functional coordination with normaltissue. These can be broadly classified into three major types.Malignant neoplasms arising from epithelial structures are calledcarcinomas, malignant neoplasms that originate from connective tissuessuch as muscle, cartilage, fat or bone are called sarcomas and malignanttumors affecting hematopoetic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. A tumor is the neoplastic growth of thedisease cancer. As used herein, a neoplasm, also referred to as a“tumor”, is intended to encompass hematopoietic neoplasms as well assolid neoplasms. Other proliferative disorders include, but are notlimited to neurofibromatosis.

The useful for inhibiting telomere elongation (such as in pharmaceuticalcompositions) provided herein are useful for modulating disease statesassociated with dysregulation of telomere length in cells. Telomerase isinvolved in multiple biological and physiological functions, including,e.g., cell proliferation and cell survival. In some embodiments, thecell proliferative disorder is associated with increased expression oractivity of telomerase or cellular growth, or both. In some embodiments,the cell proliferation is cancer.

The methods described herein are also useful for treating solid tumors(such as advanced solid tumors). In some embodiments, there is provideda method of treating lung cancer, including, for example, non-small celllung cancer (NSCLC, such as advanced NSCLC), small cell lung cancer(SCLC, such as advanced SCLC), and advanced solid tumor malignancy inthe lung. In some embodiments, there is provided a method of treatingany of ovarian cancer, head and neck cancer, gastric malignancies,melanoma (including metastatic melanoma and malignant melanoma), ovariancancer, colorectal cancer, and pancreatic cancer.

In some embodiments, the method is useful for treating one or more ofthe following: cutaneous T cell lymphoma (CTCL), leukemia, follicularlymphoma, Hodgkin lymphoma, and acute myeloid leukemia.

In some embodiments, the disease is a cancer of any one of thefollowing: basal cell carcinoma, medulloblastoma, glioblastoma, multiplemyeloma, chronic myelogenous leukemia (CML), acute myelogenous leukemia,pancreatic cancer, lung cancer (small cell lung cancer and non-smallcell lung cancer), esophageal cancer, stomach cancer, billary cancer,prostate cancer, liver cancer, hepatocellular cancer, gastrointestinalcancer, gastric cancer, and ovarian and bladder cancer. In someembodiments, the cancer is selected from the group consisting ofpancreas ductal adenocarcinoma, colon adenocarcinoma, and ovarycystadenocarcinoma. In some embodiments, the cancer is pancreas ductaladenocarcinoma. In some embodiments, the cancer is a tumor that ispoorly perfused and/or poorly vascularized.

In some embodiments, the cancer is pancreatic cancer, including forexample pancreatic adenocarcinoma, pancreatic adenosquamous carcinoma,pancreatic squamous cell carcinoma, and pancreatic giant cell carcinoma.In some embodiments, the pancreatic cancer is exocrine pancreaticcancer. In some embodiments, the pancreatic cancer is endocrinepancreatic cancer (such as islet cell carcinoma). In some embodiments,the pancreatic cancer is advanced metastatic pancreatic cancer.

Other examples of cancers that can be treated by the methods of theinvention include, but are not limited to, adenocortical carcinoma,agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-relatedlymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellarand cerebral), basal cell carcinoma, bile duct cancer (e.g.,extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignantfibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma,cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuseastrocytoma, anaplastic (malignant) astrocytoma), malignant glioma,ependymoma, oligodenglioma, meningioma, craniopharyngioma,haemangioblastomas, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, andglioblastoma), breast cancer, bronchial adenomas/carcinoids, carcinoidtumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknownprimary, central nervous system lymphoma, cervical cancer, colon cancer,colorectal cancer, chronic myeloproliferative disorders, endometrialcancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing'sfamily of tumors, eye cancer (e.g., intraocular melanoma andretinoblastoma), gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, (e.g., extracranial, extragonadal, ovarian),gestational trophoblastic tumor, head and neck cancer, hepatocellular(liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngealcancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer,laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer,liver cancer, lung cancer (e.g., small cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovariancancer, mesothelioma, metastatic squamous neck cancer, mouth cancer,multiple endocrine neoplasia syndrome, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrinecancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelialcancer, ovarian germ cell tumor, ovarian low malignant potential tumor),pancreatic cancer, parathyroid cancer, penile cancer, cancer of theperitoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor,pleuropulmonary blastoma, lymphoma, primary central nervous systemlymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer,renal cancer, renal pelvis and ureter cancer (transitional cell cancer),rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma(e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma),small intestine cancer, squamous cell cancer, testicular cancer, throatcancer, thymoma and thymic carcinoma, thyroid cancer, tuberoussclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor,and post-transplant lymphoproliferative disorder (PTLD), abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

In some embodiments, the cancer is a solid tumor (such as advanced solidtumor). Solid tumor includes, but is not limited to, sarcomas andcarcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,Kaposi's sarcoma, soft tissue sarcoma, uterine sacronomasynovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma (including for exampleadenocarcinoma, clear cell renal cell carcinoma, papillary renal cellcarcinoma, chromophobe renal cell carcinoma, collecting duct renal cellcarcinoma, granular renal cell carcinoma, mixed granular renal cellcarcinoma, renal angiomyolipomas, or spindle renal cell carcinoma),hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilm's tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is a B-cellneoplasm. Examples of B-cell neoplasms include, but are not limited to,precursor B-cell neoplasms (e.g., precursor B-lymphoblasticleukemia/lymphoma) and peripheral B-cell neoplasms (e.g., B-cell chroniclymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma(small lymphocytic (SL) NHL), lymphoplasmacytoid lymphoma/immunocytoma,mantel cell lymphoma, follicle center lymphoma, follicular lymphoma(e.g., cytologic grades: I (small cell), II (mixed small and largecell), III (large cell) and/or subtype: diffuse and predominantly smallcell type), low grade/follicular non-Hodgkin's lymphoma (NHL),intermediate grade/follicular NHL, marginal zone B-cell lymphoma (e.g.,extranodal (e.g., MALT-type+/− monocytoid B cells) and/or Nodal (e.g.,+/− monocytoid B cells)), splenic marginal zone lymphoma (e.g., +/−villous lymphocytes), Hairy cell leukemia, plasmacytoma/plasma cellmyeloma (e.g., myeloma and multiple myeloma), diffuse large B-celllymphoma (e.g., primary mediastinal (thymic) B-cell lymphoma),intermediate grade diffuse NHL, Burkitt's lymphoma, High-grade B-celllymphoma, Burkitt-like, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia).

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is a T-celland/or putative NK-cell neoplasm. Examples of T-cell and/or putativeNK-cell neoplasms include, but are not limited to, precursor T-cellneoplasm (precursor T-lymphoblastic lymphoma/leukemia) and peripheralT-cell and NK-cell neoplasms (e.g., T-cell chronic lymphocyticleukemia/prolymphocytic leukemia, and large granular lymphocyte leukemia(LGL) (e.g., T-cell type and/or NK-cell type), cutaneous T-cell lymphoma(e.g., mycosis fungoides/Sezary syndrome), primary T-cell lymphomasunspecified (e.g., cytological categories (e.g., medium-sized cell,mixed medium and large cell), large cell, lymphoepitheloid cell, subtypehepatosplenic γδ T-cell lymphoma, and subcutaneous panniculitic T-celllymphoma), angioimmunoblastic T-cell lymphoma (AILD), angiocentriclymphoma, intestinal T-cell lymphoma (e.g., +/− enteropathy associated),adult T-cell lymphoma/leukemia (ATL), anaplastic large cell lymphoma(ALCL) (e.g., CD30+, T- and null-cell types), anaplastic large-celllymphoma, and Hodgkin's lymphoma).

In some embodiments the lymphoid neoplasm (e.g., lymphoma) is Hodgkin'sdisease. For example, the Hodgkin's disease can be lymphocytepredominance, nodular sclerosis, mixed cellularity, lymphocytedepletion, and/or lymphocyte-rich.

In some embodiments, the cancer is leukemia. In some embodiments, theleukemia is chronic leukemia. Examples of chronic leukemia include, butare not limited to, chronic myelocytic I (granulocytic) leukemia,chronic myelogenous, and chronic lymphocytic leukemia (CLL). In someembodiments, the leukemia is acute leukemia. Examples of acute leukemiainclude, but are not limited to, acute lymphoblastic leukemia (ALL),acute myeloid leukemia, acute lymphocytic leukemia, and acute myelocyticleukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic,and erythroleukemia).

In some embodiments, the cancer is liquid tumor or plasmacytoma.Plasmacytoma includes, but is not limited to, myeloma. Myeloma includes,but is not limited to, an extramedullary plasmacytoma, a solitarymyeloma, and multiple myeloma. In some embodiments, the plasmacytoma ismultiple myeloma.

In some embodiments, the cancer is multiple myeloma. Examples ofmultiple myeloma include, but are not limited to, IgG multiple myeloma,IgA multiple myeloma, IgD multiple myeloma, IgE multiple myeloma, andnonsecretory multiple myeloma. In some embodiments, the multiple myelomais IgG multiple myeloma. In some embodiments, the multiple myeloma isIgA multiple myeloma. In some embodiments, the multiple myeloma is asmoldering or indolent multiple myeloma. In some embodiments, themultiple myeloma is progressive multiple myeloma. In some embodiments,multiple myeloma may be resistant to a drug, such as, but not limitedto, bortezomib, dexamethasone (Dex−), doxorubicin (Dox−), and melphalan(LR).

D. Methods for Treating Cell Proliferative Disorders

The present invention is directed to methods for inhibiting the symptomsor conditions (disabilities, impairments) associated with a cellproliferative disorder as described in detail below. As such, it is notrequired that all effects of the condition be entirely prevented orreversed, although the effects of the presently disclosed methods likelyextend to a significant therapeutic benefit for the patient. As such, atherapeutic benefit is not necessarily a complete prevention or cure fora particular condition resulting from a cell proliferative disorder, butrather, can encompass a result which includes reducing or preventing thesymptoms that result from a cell proliferative disorder, reducing orpreventing the occurrence of such symptoms (either quantitatively orqualitatively), reducing the severity of such symptoms or physiologicaleffects thereof, and/or enhancing the recovery of the individual afterexperiencing a cell proliferative disorder symptoms.

Specifically, a composition of the present invention (such as any of thecompounds useful for inhibiting telomere elongation disclosed herein),when administered to an individual, can treat or prevent one or more ofthe symptoms or conditions associated with a cell proliferative disorderand/or reduce or alleviate symptoms of or conditions associated withthis disorder. As such, protecting an individual from the effects orsymptoms resulting from an a cell proliferative disorder includes bothpreventing or reducing the occurrence and/or severity of the effects ofthe disorder and treating a patient in which the effects of the disorderare already occurring or beginning to occur. A beneficial effect caneasily be assessed by one of ordinary skill in the art and/or by atrained clinician who is treating the patient. Preferably, there is apositive or beneficial difference in the severity or occurrence of atleast one clinical or biological score, value, or measure used toevaluate such patients in those who have been treated with the methodsof the present invention as compared to those that have not.

The methods can be practiced in an adjuvant setting. “Adjuvant setting”refers to a clinical setting in which an individual has had a history ofa proliferative disease, particularly cancer, and generally (but notnecessarily) been responsive to therapy, which includes, but is notlimited to, surgery (such as surgical resection), radiotherapy, andchemotherapy. However, because of their history of the proliferativedisease (such as cancer), these individuals are considered at risk ofdevelopment of the disease. Treatment or administration in the “adjuvantsetting” refers to a subsequent mode of treatment. The degree of risk(i.e., when an individual in the adjuvant setting is considered as “highrisk” or “low risk”) depends upon several factors, most usually theextent of disease when first treated.

The methods provided herein can also be practiced in a “neoadjuvantsetting,” i.e., the method can be carried out before theprimary/definitive therapy. In some embodiments, the individual haspreviously been treated. In some embodiments, the individual has notpreviously been treated. In some embodiments, the treatment is a firstline therapy.

In some aspects, provided herein are methods for treating a cellproliferative disorder in an individual by administering an effectiveamount of any of the compounds (such as in pharmaceutical compositions)disclosed herein. In some embodiments, the cell proliferative disease iscancer, such as metastatic cancer. In other embodiments the cancer is acancer of the skin, connective tissue, adipose, breast, lung, liver,stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon,colorectal, prostate, central nervous system (CNS), brain, retina, ahematologic tumors (such as myeloma, leukemia and lymphoma), or any ofthe cancers disclosed herein. The compound or composition of the presentinvention may be administered orally, intra-arterially, intranasal,intraperitoneally, intravenously, intramuscularly, subcutaneously, ortransdermally. In some embodiments, the individual is a human.

E. Administration of Compounds Useful for Inhibiting Telomere Elongation

In some embodiments, the compounds useful for inhibiting telomereelongation disclosed herein (such as in a pharmaceutical compositions)are administered in the form of an injection. The injection can comprisethe compound in combination with an aqueous injectable excipient orcarrier. Non-limiting examples of suitable aqueous injectable excipientsor carriers are well known to persons of ordinary skill in the art, andthey, and the methods of formulating the formulations, may be found insuch standard references as Alfonso A R: Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton Pa., 1985. Suitableaqueous injectable excipients or carriers include water, aqueous salinesolution, aqueous dextrose solution, and the like, optionally containingdissolution enhancers such as 10% mannitol or other sugars, 10% glycine,or other amino acids. The composition can be injected subcutaneously,intraperitoneally, or intravenously.

In some embodiments, intravenous administration is used, and it can becontinuous intravenous infusion over a period of a few minutes to anhour or more, such as around fifteen minutes. The amount administeredcan vary widely depending on the type of antisense oligonucleotide, sizeof a unit dosage, kind of excipients or carriers, and other factors wellknown to those of ordinary skill in the art. The antisenseoligonucleotide can comprise, for example, from about 0.001% to about10% (w/w), from about 0.01% to about 1%, from about 0.1% to about 0.8%,or any range therein, with the remainder comprising the excipient(s) orcarrier(s).

For oral administration, the compound useful for inhibiting telomereelongation can take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients or carriers such as binding agents; fillers; lubricants;disintegrants; or wetting agents. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups orsuspensions, or they can be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations can also contain buffer salts, flavoring, and coloring asappropriate.

In some embodiments, the compound useful for inhibiting telomereelongation (such as any of the compounds disclosed herein) can beadministered by inhalation through an aerosol spray or a nebulizer thatcan include a suitable propellant such as, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or a combination thereof. Inone non-limiting example, a dosage unit for a pressurized aerosol can bedelivered through a metering valve. In another embodiment, capsules andcartridges of gelatin, for example, can be used in an inhaler and can beformulated to contain a powderized mix of the compound with a suitablepowder base such as, for example, starch or lactose.

In some embodiments, the amount of the compound useful for inhibitingtelomere elongation in the composition (such as a pharmaceuticalcomposition) is included in any of the following ranges: about 0.5 toabout 5 mg, about 5 to about 10 mg, about 10 to about 15 mg, about 15 toabout 20 mg, about 20 to about 25 mg, about 20 to about 50 mg, about 25to about 50 mg, about 50 to about 75 mg, about 50 to about 100 mg, about75 to about 100 mg, about 100 to about 125 mg, about 125 to about 150mg, about 150 to about 175 mg, about 175 to about 200 mg, about 200 toabout 225 mg, about 225 to about 250 mg, about 250 to about 300 mg,about 300 to about 350 mg, about 350 to about 400 mg, about 400 to about450 mg, or about 450 to about 500 mg. In some embodiments, the amount ofthe compounds useful for inhibiting telomere elongation in the effectiveamount of the pharmaceutical composition (e.g., a unit dosage form) isin the range of about 5 mg to about 500 mg, such as about 1 mg to about10 mg, 1 mg to about 15 mg, 1 mg to about 20 mg, 1 mg to about 25 mg, 1mg to about 30 mg, 10 mg to about 20 mg, 10 mg to about 30 mg, 10 mg toabout 40 mg, 20 mg to about 30 mg, 30 mg to about 40 mg, 30 mg to about300 mg or about 50 mg to about 200 mg. In some embodiments, theconcentration of the compound useful for inhibiting telomere elongationin the pharmaceutical composition is dilute (about 0.1 mg/ml) orconcentrated (about 100 mg/ml), including for example any of about 0.1to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 to about 10mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6 mg/ml, about 5mg/ml. In some embodiments, the concentration of the compound useful forinhibiting telomere elongation is at least about any of 0.5 mg/ml, 1.3mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml,8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40mg/ml, or 50 mg/ml.

Exemplary effective amounts of a compound useful for inhibiting telomereelongation in the pharmaceutical composition include, but are notlimited to, at least about any of 5 mg/m², 10 mg/m², 15 mg/m², 20 mg/m²,25 mg/m², 30 mg/m², 50 mg/m², 60 mg/m², 75 mg/m², 80 mg/m², 90 mg/m²,100 mg/m², 120 mg/m², 125 mg/m², 150 mg/m², 160 mg/m², 175 mg/m², 180mg/m², 200 mg/m², 210 mg/m², 220 mg/m², 250 mg/m², 260 mg/m², 300 mg/m²,350 mg/m², 400 mg/m², 500 mg/m², 540 mg/m², 750 mg/m², 1000 mg/m², or1080 mg/m². In various embodiments, the pharmaceutical compositionincludes less than about any of 350 mg/m², 300 mg/m², 250 mg/m², 200mg/m², 150 mg/m², 120 mg/m², 100 mg/m², 90 mg/m², 50 mg/m², or 30 mg/m²of a compound useful for inhibiting telomere elongation (such as any ofthe compounds disclosed herein). In some embodiments, the amount of thecompound useful for inhibiting telomere elongation per administration isless than about any of 25 mg/m², 22 mg/m², 20 mg/m², 18 mg/m², 15 mg/m²,14 mg/m², 13 mg/m², 12 mg/m², 11 mg/m², 10 mg/m², 9 mg/m², 8 mg/m², 7mg/m², 6 mg/m², 5 mg/m², 4 mg/m², 3 mg/m², 2 mg/m², or 1 mg/m². In someembodiments, the effective amount of a compound useful for inhibitingtelomere elongation in the pharmaceutical composition is included in anyof the following ranges: about 1 to about 5 mg/m², about 5 to about 10mg/m², about 10 to about 25 mg/m², about 25 to about 50 mg/m², about 50to about 75 mg/m², about 75 to about 100 mg/m², about 100 to about 125mg/m², about 125 to about 150 mg/m², about 150 to about 175 mg/m², about175 to about 200 mg/m², about 200 to about 225 mg/m², about 225 to about250 mg/m², about 250 to about 300 mg/m², about 300 to about 350 mg/m²,or about 350 to about 400 mg/m². In some embodiments, the effectiveamount of a compound useful for inhibiting telomere elongation in thepharmaceutical composition is about 5 to about 300 mg/m², such as about20 to about 300 mg/m², about 50 to about 250 mg/m², about 100 to about150 mg/m², about 120 mg/m², about 130 mg/m², or about 140 mg/m², orabout 260 mg/m².

In some embodiments of any of the above aspects, the effective amount ofa compound useful for inhibiting telomere elongation in thepharmaceutical composition includes at least about any of 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38mg/kg, 39 mg/kg, or 40 mg/kg or more. In various embodiments, theeffective amount of a compound useful for inhibiting telomere elongation(such as any of the compounds disclosed herein) in the pharmaceuticalcomposition includes less than about any of 350 mg/kg, 300 mg/kg, 250mg/kg, 200 mg/kg, 150 mg/kg, 100 mg/kg, 50 mg/kg, 30 mg/kg, 25 mg/kg, 20mg/kg, 10 mg/kg, 7.5 mg/kg, 6.5 mg/kg, 5 mg/kg, 3.5 mg/kg, 2.5 mg/kg, or1 mg/kg of a compound useful for inhibiting telomere elongation.

Exemplary dosing frequencies for the pharmaceutical compositions (suchas a pharmaceutical composition containing any of the compounds usefulfor inhibiting telomere elongation disclosed herein) include, but arenot limited to, daily; every other day; twice per week; three times perweek; weekly without break; weekly, three out of four weeks; once everythree weeks; once every two weeks; weekly, two out of three weeks. Insome embodiments, the pharmaceutical composition is administered aboutonce every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6weeks, or once every 8 weeks. In some embodiments, the composition isadministered at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week, or three times daily, two times daily. In someembodiments, the intervals between each administration are less thanabout any of 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or1 day. In some embodiments, the intervals between each administrationare more than about any of 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 8 months, or 12 months. In some embodiments, there isno break in the dosing schedule. In some embodiments, the intervalbetween each administration is no more than about a week.

The administration of the pharmaceutical composition can be extendedover an extended period of time, such as from about a month up to aboutseven years. In some embodiments, the composition is administered over aperiod of at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18,24, 30, 36, 48, 60, 72, or 84 months.

V. Kits

The present disclosure provides a pharmaceutical pack or kit comprisingone or more containers comprising a compound of Formulae (I)-(IX) usefulfor the treatment or prevention of cancer. The kit can further compriseinstructions for use in the treatment of cancer.

The present disclosure also provides a pharmaceutical pack or kitcomprising one or more containers comprising one or more of theingredients of the pharmaceutical compositions of the presentembodiments. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

VI. Specific Embodiments of the Invention

The compositions and methods described herein inhibit the extension oftelomeres by telomerase. The compounds and methods also inhibitproliferation of cancer cells.

It is believed that the compounds described herein are recognized by thetelomerase enzyme and incorporated into the growing telomere chain. Oncethe compound is incorporated into the telomere chain, the telomeraseenzyme is unable to add addition nucleotides to the telomere chain. Inthis way the telomere chain is stopped or capped and further elongationof the telomere is inhibited.

It is preferred that the compounds described herein are preferentiallyaccepted and used by the telomerase enzyme and are not used by mammalianDNA or RNA polymerases. The compounds inhibit the elongation oftelomeres by telomerase as least 2× better than inhibiting theelongation of DNA or RNA by DNA or RNA polymerases, at least 5× betterthan, at least 10× better than inhibiting the elongation of DNA or RNAby DNA or RNA polymerases.

In accordance with this invention, compounds are provided having formula(VIII)

wherein G is selected from guanine-9-yl, or its 1-deaza or 3-deazaanalogs, Y independently is —OH, —NH(CH₂)_(n)NH(CH₂)_(n)NHR³; or—N[(CH₂)NH₂](CH₂)_(n)NHR³; R³ is —H or —(CH₂)_(n)NH₂; n independently is2-4; with the proviso that at least one Y is—NH(CH₂)_(n)NH(CH₂)_(n)NHR³; or —N[(CH₂)_(n)NH₂](CH₂)_(n)NHR³; and thesalts, hydrates, tautomers and solvates thereof. In one embodiment G isguanine-9-yl.

In one embodiment, at least one Y is —NH(CH₂)NH(CH₂)_(n)NHR³, R³ is —Hor —(CH₂)_(n)NH₂ and n independently is 2-4.

In one embodiment, at least one Y is —NH(CH₂)₃NH(CH₂)₄NH₂ or—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂. In one embodiment one Y is—NH(CH₂)₃NH(CH₂)₄NH₂ and the other Y is —OH. In one embodiment one Y is—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ and the other Y is —OH. In one embodiment,both Ys are —NH(CH₂)₃NH(CH₂)₄NH₂. In one embodiment both Ys are—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂.

In one embodiment, at least one Y is —N[(CH₂)_(n)NH₂](CH₂)_(n)NHR³, R³is —H or —(CH₂), NH₂ and n independently is 2-4.

In one embodiment, the compound of formula VIII is the enriched orisolated (R) enantiomer. In another embodiment, the compound of formulaVIII is the enriched or isolated (S) enantiomer.

In accordance with this invention, pharmaceutical compositions areprovided comprising the compounds of Formula VIII with apharmaceutically acceptable excipient.

In accordance with this invention, methods are provided for inhibitingtelomere elongation comprising contacting a cell with the compounds ofFormula VIII of this invention or the pharmaceutical compositions ofthis invention. In some embodiments, the cell is a cancer cell.

In accordance with this invention, methods are provided for shorteningtelomere length in a cell or tissue comprising contacting the cell ortissue with the compounds of Formula VIII of this invention or thepharmaceutical compositions of this invention.

In accordance with this invention, methods are provided for treatingcancer in a patient by administering an effective amount of thecompounds of Formula VIII of this invention or the pharmaceuticalcompositions of this invention to the patient. In one embodiment, thecancer is metastatic cancer. In one embodiment, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma).

In accordance with this invention, methods are provided for treating apatient by administering an effective amount of the compounds of FormulaVIII of this invention or the pharmaceutical compositions of thisinvention wherein the method involves oral, intra-arterial, intranasal,intraperitoneal, intravenous, intramuscular, subcutaneous, ortransdermal administration of the compound or the pharmaceuticalcompositions of the invention.

In accordance with this invention, methods are provided using compoundshaving formula (IX):

wherein G is selected from guanine-9-yl, or its 1-deaza or 3-deazaanalogs, X independently is —OH, a monoposphate, a diphosphate, or—OCH(R¹)OC(O)OR¹, R¹ independently is —H, or C₁-C₅ alkyl; and the salts,hydrates, tautomers and solvates thereof; under conditions whereintelomere elongation is inhibited. In one embodiment, the compounds ofFormula IX wherein at least one X is —OCH₂OC(O)OR¹ and R¹ is C₁-C₅alkyl. In one embodiment, the compounds of Formula VIII wherein one X is—OH and the other X is —OCH₂OC(O)OR¹ and R¹ is C₁-C₅ alkyl. In oneembodiment, the compounds of Formula IX wherein one X is —OH and theother X is —OCH₂OC(O)OCH(CH₃)₂. In another embodiment both Xs are—OCH₂OC(O)OCH(CH₃)₂. In another embodiment, one X is —OH and the other Xis diphosphate. A specific compound of Formula IX is9-[2-(phosphonomethoxy)propyl]-guanine diphosphate; PMPGpp, or apharmaceutically acceptable salt thereof. A specific compound of FormulaIX is (R)-9-[2-(phosphonomethoxy)propyl]-guanine diphosphate;(R)-PMPGpp, or a pharmaceutically acceptable salt thereof. A specificcompound of Formula IX is (S)-9-[2-(phosphonomethoxy)propyl]-guaninediphosphate; (S)-PMPGpp, or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula IX is the enriched orisolated (R) enantiomer. In another embodiment, the compound of FormulaIX is enriched or isolated (S) enantiomer.

In accordance with this invention, methods are provided for inhibitingtelomere elongation comprising contacting a cell with the compounds ofFormula IX of this invention or the pharmaceutical compositions of thisinvention. In some embodiments, the cell is a cancer cell.

In accordance with this invention, methods are provided for shorteningtelomere length in a cell or tissue comprising contacting the cell ortissue with the compounds of Formula IX of this invention or thepharmaceutical compositions of this invention.

In accordance with this invention, methods are provided for treatingcancer in a patient by administering an effective amount of thecompounds of Formula IX of this invention or the pharmaceuticalcompositions of this invention to the patient. In one embodiment, thecancer is metastatic cancer. In one embodiment, the cancer is a cancerof the skin, connective tissue, adipose, breast, lung, liver, stomach,pancreas, ovary, cervix, uterus, kidney, bladder, colon, colorectal,prostate, central nervous system (CNS), brain, retina and hematologictumors (such as myeloma, leukemia and lymphoma).

In accordance with this invention, methods are provided for treating apatient by administering an effective amount of the compounds of FormulaIX of this invention or the pharmaceutical compositions of thisinvention wherein the method involves oral, intra-arterial, intranasal,intraperitoneal, intravenous, intramuscular, subcutaneous, ortransdermal administration of the compound or the pharmaceuticalcompositions of the invention.

Use of the compounds of Formula IX in medicine.

Use of the compounds of Formula VIII and Formula IX for treating cancer.

The compounds of the invention inhibit the elongation or extension oftelomeres in cells by telomerase, including cancer cells, the resultanteffect of which is to inhibit proliferation of the cells. Accordingly, aprimary application of the compounds of the invention is as cancertherapeutics, and the invention provides pharmaceutical formulations ofthe compounds that may be utilized in this manner.

Exemplary compounds of the invention include those depicted in Table 1using Formula VIII below:

TABLE 1 G *Enantiomer Y¹ Y² Guanine- Mixed —OH —NH(CH₂)₃NH(CH₂)₄NH₂•9-yl Guanine- Mixed —NH(CH₂)₃NH(CH₂)₄NH₂ —NH(CH₂)₃NH(CH₂)₄NH₂ 9-ylGuanine- (R) —OH —NH(CH₂)₃NH(CH₂)₄NH₂• 9-yl Guanine- (R)—NH(CH₂)₃NH(CH₂)₄NH₂ —NH(CH₂)₃NH(CH₂)₄NH₂ 9-yl Guanine- (S) —OH—NH(CH₂)₃NH(CH₂)₄NH₂• 9-yl Guanine- (S) —NH(CH₂)₃NH(CH₂)₄NH₂—NH(CH₂)₃NH(CH₂)₄NH₂ 9-yl Guanine- Mixed —OH—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- Mixed—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ —OH 9-yl Guanine- (R) —OH—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- (R)—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ —OH 9-yl Guanine- (S) —OH—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- (S)—NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂ —OH 9-yl Guanine- Mixed —OH—NH[(CH₂)₃NH₂](CH₂)₄NH₂ 9-yl Guanine- Mixed —NH[(CH₂)₃NH₂](CH₂)₄NH₂—NH[(CH₂)₃NH₂](CH₂)₄NH₂ 9-yl Guanine- (R) —OH —NH[(CH₂)₃NH₂](CH₂)₄NH₂9-yl Guanine- (R) —NH[(CH₂)₃NH₂](CH₂)₄NH₂ —NH[(CH₂)₃NH₂](CH₂)₄NH₂ 9-ylGuanine- (S) —OH —NH[(CH₂)₃NH₂](CH₂)₄NH₂ 9-yl Guanine- (S)—NH[(CH₂)₃NH₂](CH₂)₄NH₂ —NH[(CH₂)₃NH₂](CH₂)₄NH₂ 9-yl Guanine- Mixed —OH—NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- Mixed—NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ —NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-ylGuanine- (R) —OH —NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- (R)—NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ —NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-ylGuanine- (S) —OH —NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-yl Guanine- (S)—NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ —NH[(CH₂)₃NH₂](CH₂)₄NH(CH₂)₃NH₂ 9-yl

Methods are provided using compounds which include those depicted inTable 2 using Formula IX below:

TABLE 2 G *Enantiomer X¹ X² Guanine- Mixed —OH —OCH₂OC(O)OCH(CH₃)₂• 9-ylGuanine- Mixed —OCH₂OC(O)OCH(CH₃)₂ —OCH₂OC(O)OCH(CH₃)₂ 9-yl Guanine- (R)—OH —OCH₂OC(O)OCH(CH₃)₂• 9-yl Guanine- (R) —OCH₂OC(O)OCH(CH₃)₂—OCH₂OC(O)OCH(CH₃)₂ 9-yl Guanine- (S) —OH —OCH₂OC(O)OCH(CH₃)₂• 9-ylGuanine- (S) —OCH₂OC(O)OCH(CH₃)₂ —OCH₂OC(O)OCH(CH₃)₂ 9-yl Guanine- Mixed—OH Diphosphate 9-yl Guanine- (R) —OH Diphosphate 9-yl Guanine- (S) —OHDiphosphate 9-yl

EXAMPLES

The following Examples illustrate the synthesis and activities ofcompounds of the invention.

Example 1. Synthesis of Compounds A. Synthesis of R-(((Guanine-9 yl)propan-2-oxy) methyl) phosphonic acid diphosphate

The scheme for synthesizing R-(((Guanine-9-yl) propan-2-oxy) methyl)phosphonic acid diphosphate is shown in the scheme below.

Step A:

20 mmol 2-amino-6-chloropurine (Aldrich 34,230-0) was co-evaporated withdry toluene three (3) times. 150 ml dry DMF, 30 mmol R-(+)-propylenecarbonate (Aldrich 54,001-3), 20 mmol dry potassium carbonate was addedand the mixture was heated at 90° C. for 24 hrs. After cooling, 100 mlmethanol was added and the reaction mixture was cooled to 0° C. Thesolid was filtered and washed with 50 ml methanol. The pH of thefiltrate was adjusted to pH7 with acetic acid; then it was evaporated todryness. The product was purified using Silica gel flash chromatographystarting with 5% methanol in dichloromethane (DCM) followed by 10%methanol. The yield on average was 50% based upon2-amino-6-chloropurine.

Step B:

2 mmol of the 2-amino-6-chloro acyclic compound was co evaporated withdry acetonitrile and dissolved in 10 ml N-methyl-2-pyrrolidone (NMP). 6mmol Lithium-tert-butoxide and 3 mmol of phosphonylating reagent (SantaCruz Biotechnology sc-211323) was added and the reaction mixture heatedat 70° C. for 5 hrs. The progress of the reaction was followed by HPLC(17.3 min). After cooling, water was added and the pH of the solutionwas adjusted to pH7. The product was extracted with methylene chlorideand evaporated to 10 ml (NMP). This solution was used in the next step.

Step C:

The NMP solution was co-evaporated with acetonitrile three (3) times,then 7 mmol sodium bromide was added and the reaction mixture was cooledto 0° C. 10.6 mmol trimethylsilyl chloride (TMS-Cl) was added and thereaction mixture was heated at 75° C. for 5 hrs. The reaction wasfollowed by HPLC (11.6 min). When complete, the reaction mixture wascooled, water was added and extracted with ethyl acetate. The pH of thewater phase was adjusted to pH 3-4 with NaOH and evaporated to about 10ml N-methyl-2-pyrrolidone (NMP). 50 ml ethanol was added and aftercooling the salt was filtered out. The solution was evaporated toN-methyl-2-pyrrolidone (NMP).

Step D:

The NMP solution from the previous step was diluted with 20 ml ethanoland 20 ml 5M HCl was added. This solution was kept at 55° C. for 3 hrs,the reaction was monitored by HPLC (9.5 min), cooled, and 20 ml ethanolwas added. The precipitated salt was filtered off and the solution wasevaporated. The remaining NMP was removed by trituration withacetonitrile and the end product filtered.

HPLC method: Solvent A: 50 mM triethylammonium acetate; Solvent B:acetonitrile; Gradient: 0-50% B in 25 min.: Flow rate 1 ml/min; C₁₈column 4.6×250 mm; Steps A-C monitored at 300-330 nm, Step D monitoredat 260 nm.

B. Synthesis of S-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acidDiphosphate

The scheme for synthesizing S-(((Guanine-9-yl) propan-2-oxy) methyl)phosphonic acid diphosphate is the same as described forR-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate asshown in Example 1A above substituting S-(−)-propylene carbonate forR-(+)-propylene carbonate.

C. Synthesis of R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acidDiisopropyloxy Ester

The synthesis method is shown in the scheme below.

In the scheme above, 0.5 mmol R-(((Guanine-9-yl) propan-2-oxy) methyl)phosphonic acid was co-evaporated with dry acetonitrile, then dissolvedin 5 ml of NMP. 280 μl triethylamine (4 eq) and 160 mgtetrabutylammonium bromide (1 eq) was added and the mixture heated at50° C. At that temperature 380 μl (5 eq) chloromethyl isopropylcarbonate (Santa Cruz Biotech sc-211074) was added and the reactionmixture stirred at 50° C. for 5 hrs (or less, depending on the HPLCmonitoring). The product elutes at 22.8 min and the monoester at 14 min.After the reaction was complete, the mixture was cooled down and theproducts precipitated with cyclohexane. To the oily residue methylenechloride was added and extracted with water. The organic layer was driedand evaporated to oil containing a small amount of NMP. After dilutionwith water the solution was purified by HPLC. The product was extractedfrom collected fractions with methylene chloride and the solutionevaporated. The resulting oil was dissolved in DMSO and theconcentration determined by UV spectrophotometry.

HPLC method: Solvent A: 50 mM triethylammonium acetate; Solvent B:acetonitrile; Gradient: 0-50% B in 25 min.; Flow rate 1 ml/min; C₁₈column 4.6×250 mm; Steps A-C monitored at 300-330 nm; Step D monitoredat 260 nm.

Synthesis of S-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic aciddisoproxil was done following the same procedure beginning withS-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid.

D. Synthesis of R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acidSpermine or Spermidine Amidate

The synthesis method is shown in the scheme below.

In the scheme above, 0.1 mmol R-(((Guanine-9-yl) propan-2-oxy) methyl)phosphonic acid was co evaporated with dry DMF, then dissolved in 5 mlof DMF. 80 mg carbonyldiimidazole (5 eq) was added and the mixturestirred at room temperature for 24 hrs. then 30 μl methanol was addedand the reaction mixture stirred for 10 min. 5 eq of spermine orspermidine was added and the reaction mixture stirred 24 hrs. After theaddition of 5 ml 50 mM triethylammonium acetate buffer, the reactionmixture was evaporated and dissolved in water for HPLC purification. Theproducts eluted later, then the starting material and were collected asa set of peaks (primary or secondary amine reacted). The products werenot retained on ion exchange column and the ³¹P phosphorous NMR spectrumshowed peaks at 19.946 and 19.873 ppm. The starting material has asingle peak at 16.258 ppm.

HPLC method: Solvent A: 50 mM triethylammonium acetate; Solvent B:acetonitrile: Gradient: 0-50% B in 25 min.: Flow rate 1 ml/min; C₁₈column 4.6×250 mm: Steps A-C monitored at 300-330 nm; Step D monitoredat 260 nm.

Synthesis of S-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acidspermine or spermidine amidate is done by following the above procedurestarting with S-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid.

E. Synthesis of(((2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methyl)allyl)oxy)methyl)phosphonicacid

The synthesis method is shown in the scheme below.

Step 1: Preparation of Compound 2: To a suspension of paraformaldehyde(3.65 g, 120 mmol) in diisopropylphosphonate (20 g, 120 mmol) was addedtriethylamine (1.66 ml). The reactants were heated in a 130° C. oil bathwith vigorous stirring for 4 hours. The volatiles were removed undervacuum and the residue was chromatographed on silica gel using ethylacetate to afford Compound 2 (20 g, yield: 85%)

Step 2: Preparation of Compound 3: A solution of Compound 2 (2.62 g, 13mmol) in DMF (10 mL) was cooled to −78° C. and NaH (1.08 g) was added inportions. The mixture was slowly warmed to −20° C. and2-chloromethyl-3-chloropropene (5 g, 40 mmol) was added. The reactionmixture was stirred at room temperature for 6 hours, partitioned betweenethyl acetate and water, and the organic layer was evaporated. Theresidue was chromatographed on silica gel using ethyl acetate:petroleumether 1:1 to achieve 3 (0.65 g, yield: 18%)

Step 3: Preparation of Compound 4: Compound 3 (28.4 mg, 0.1 mmol) wasdissolved in DMF (1 mL), 2-amino-6-chloro purine (17 mg, 0.1 mmol) andfresh Cs₂CO₃ (32.5 mg, 1 mmol) was added to the solution and withvigorous stirring overnight at room temperature. The mixture was dilutedwith ethyl acetate and filtered to remove the solid. The aqueous layerwas extracted twice with ethyl acetate, the organic layers were combinedand washed with brine, dried over Na₂SO₄ and concentrated under vacuum.The residue was purified by flash column chromatography (ethylacetate/methanol 9:1) to achieve Compound 4 (10 mg, yield: 30%)

Step 4: Preparation of Compound 6: Compound 4 (500 mg, 1.2 mmol) wasdissolved in trifluoroacetic acid (TFA) (5 mL). The resulting mixturewas stirred at room temperature for 24 hours under argon until all thestarting material was consumed. The reaction mixture was concentratedunder reduced pressure to give Compound 6 as brown oil (480 mg, 100%yield, LCMS confirmed). Compound 6 was used to the next step withoutfurther purification.

Step 5: Preparation of Compound 7: To a solution of Compound 6 (480 mg,crude) in acetonitrile (MeCN) (5 ml), was added bromotrimethylsilane(TMSBr) (2 mL) dropwisely at room temperature under argon. The resultingmixture was stirred at room temperature for 36 hours. LCMS showed 85%product formed and 13% starting material still existed. Therefore thereaction mixture was stirred at room temperature until the startingmaterial was consumed.

F. Synthesis of Acyclic Nucleoside Phosphonate Polyamine Conjugates

The synthesis method is shown in the scheme below.

Method A: The acyclic nucleoside phosphonate free acid (0.1 mmol) wasdried either by co-evaporation with dry DMF or in desiccator overphosphorous pentoxide. The compound was dissolved in 5 mL dry DMF and0.5 mmol carbonyldiimidazole was added then the reaction mixture stirredat room temperature overnight. After the addition of 30 μL dry methanol,0.5 mmol of polyamine was added and the reaction mixture stirred atambient temperature overnight. The solvents were removed by evaporationin vacuo and the residue triturated with diethyl ether then dissolved inwater. The product was isolated by HPLC using a C18 reversed phasecolumn with a linear gradient of acetonitrile to 40% in 30 min and 50 mMtriethylammonium acetate as eluent A.

Method B: Dry acyclic nucleoside phosphonate free acid—0.1 mmol—wasdissolved in 4 mL DMF and 1 mL pyridine and 60 mg dimethylaminopyridine(DMAP) was added. 1 mmol Triphenylphosphine was dissolved in 1.5 mL DMF,and 1 mmol pyridinium disulfide was dissolved also in 1.5 mL DMF. Thetwo solutions were added to the phosphonate solution simultaneously. Thereaction mixture was warmed to a maximum of 50° C. for 10 minutes todissolve, then stirred for 4 hours at room temperature. The reactionmixture was poured into 100 mL acetone containing 2 mL saturated lithiumperchlorate and centrifuged. The pellet was then treated with 10 mmolpolyamine in 3 mL of water for one hour. The product wasisolated/purified as above.

PMPG spermine conjugate: ³¹P NMR 19.786 ppm, MS m/z MH⁺ 488.5

PMPG spermidine conjugate: ³¹P NMR 20.091 ppm, MS m/z MH⁺ 431.2

PMIBeG spermidine conjugate: ³¹P NMR 19.476 ppm, MS m/z MH⁺ 443.3

PMPG decylamine conjugate: ³¹P NMR 18.572 ppm, MS m/z MH⁺ 443.4

G. Synthesis of Acyclic Nucleoside Phosphonate Polyamine Conjugates

The synthesis method is shown in the scheme below.

Method C: Dry acyclic nucleoside free acid—0.1 mmol—was dissolved in 6mL of DMF/pyridine 1:1 and 0.5 mmol polyamine and 10 mmoldicyclohexylcarbodiimide was added. The mixture was stirred at 85° C.for 4 hours. After cooling, the reaction mixture was evaporated invacuo, triturated with ether, then suspended in acetone and centrifuged.The pellet was taken up in water and purified by HPLC as above using asteeper gradient, up to 70% acetonitrile. In this reaction, the bissubstituted compound forms as well and elutes later, than the monosubstituted compound.

Bis PMPG spermine: ³¹P NMR 20.014 ppm, MS m/z MH⁺ 773.4

Example 2. Activity of Compounds in Biochemical and Cell-Based Assays

Materials and Methods

A. Primer Extension Assay

(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate(PMPGpp) acts as a chain terminator and competes with dGTP.

Purified human telomerase was incubated with d(TTAGGG)₃ (SEQ ID NO: 1)and deoxy-nucleoside triphosphates for 90 minutes at 37° C. Allreactions were done in the presence of 200 dTTP and 10 μM dATP (50,000cpm/pmol a-³³P-dATP). Activity was determined by incubating affinitypurified telomerase extract with 1 μM primer [d(TTAGGG)₃] (SEQ ID NO:1), 200 μM dTTP, 50 μM dGTP, 10 μM dATP, 10 μCi [α-³³P]dATP (2000-4000Ci/mmol) in a buffer containing 50 mM (N-2-hydroxyethyl piperazine-N′-3propanesulfonic acid)-NaOH pH 8.5, 1 mM MgCl₂, 1 mM DTT, 5% glycerol,0.5 mM EGTA and 100 mM KOAc in a final reaction volume of 40 μl. Primerextension products were analyzed on a 15% polyacrylamide gel containing7 M urea. A characteristic telomerase ladder has multiple bands. If acompound is not a substrate then (TTAGGG)₃TTA (21 nt) (SEQ ID NO: 2). Ifa compound is a chain terminator then (TTAGGG)₃TTAG* (22 nt) (SEQ ID NO:3).

FIG. 1 is a photograph of a 15% polyacrylamide gel containing 7 M ureashowing the primer extension products in the presence of compounds.Reactions containing 50 or 100 μM dGTP, (lanes 1 and 2) show acharacteristic 6 nucleotide ladder. Lanes 8-11 contain increasingconcentrations of PMPGpp (ID#142692) in addition to 50 μM dGTP. Lane 12contains only 50 μM PMPGpp (ID#142692). Lanes 3-7 show a comparisonusing the known chain terminator, 3′-azido-dGTP.

IC 50s for (R)-PMPGpp and (S)-PMPGpp are shown in Table 3.(R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphateis efficiently recognized by human telomerase and added to the 3′ end ofa telomeric primer resulting in chain termination. The molecule competeswith dGTP and when measured in a cell free assay, using purifiedtelomerase and dGTP concentrations of 50 μM, shows an IC50 of about 1.0μM.

B. Flash Plate Assay

Determining IC50 for PMPGpp, (R)-PMPGpp and (S)-PMPGpp. Telomeraseactivity was determined using the “flash-plate” assay withconcentrations of PMPGpp ranging from 0.05 μM to 50 μM. The results areshown in Table 3.

Compound was pre-incubated with affinity purified telomerase extract,100 nM primer [5′ biotin-d(AATCCGTCGAGCAGAGTT)] (SEQ ID NO: 4), 120 μMdTTP, 20 μM dGTP, 10 dATP, in a buffer containing 50 mM Tris-Acetate (pH8.2), 1 mM MgCl₂, 1 mM DTT, 0.5 mM EGTA and 150 mM KOAc, 5.4 nM³³P-labeled [d(CCCTAACCCTAACCCTAACCC)] (SEQ ID NO: 5 (6×10⁹ cpm/nmol) ina final reaction volume of 30 μl. Incubations were done for 90 min at37° C. Following stoppage of the reaction with EDTA (10 mM), thereaction products were captured in a streptavidin coated 96 wellFlashPlate. After a 2 hr capture period for the primer, the plates werewashed five times (2×SSC, 0.1% SDS, 10 mM EDTA) and counted.

TABLE 3 IC50 of Compounds in Flashplate and Primer Extension AssayPrimer Extension Compound Flashplate IC 50 IC50 (R)-PMPGpp  1.0 μM  1.0μM (S)-PMPGpp 14.1 μM 13.5 μM

C. Ex-Vivo Assay

Telomerase inhibition is observed in cells following the addition of theprodrugs (R)-PMPG-diisopropyloxy ester or (S)-PMPG-diisopropyloxy ester.HT3 cells were treated for three days with increasing concentrations ofPMPG-diisopropyloxy ester ranging from 0.2 μM to 20 μM. Cells (HT3RPMI+10% PBS) were incubated with compound for 24 hr after which PSTSprimer (phosphorothioate-d(AATCCGTCGAGCAGAGTT (SEQ ID NO: 6) containinga 5′ Cy-5 label) (7.5 μM) and fresh compound are added for an additional24 hr. Cells were lysed and endogenous telomerase inactivated by heating(15 min, 75° C.). Products were amplified by 30 cycles of PCR under TRAPconditions set forth above and quantified. The results of twoindependent experiments are shown in Table 4.

TABLE 4 IC50 of Compounds in Ex vivo assay Compound Ex vivo Assay IC 50(R)-PMPG diisopropyloxy ester 1.2-3.2 μM (S)-PMPG diisopropyloxyester >20 μM

A prodrug, (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic aciddiisopropyloxy ester inhibited telomerase in a cell based telomeraseassay, having an IC50 value of between 1.0-3.0 μM. The prodrug itselfwas inactive in the cell free telomerase assay indicating that it isconverted to the active molecule in cells.

D. Telomere Repeat Fragment Assay

Addition of PMPG diisopropyloxy ester prodrug to cells results intelomere shortening. Three different cancer cell lines were treated witheither 10 μM or 20 μM PMPG diisopropyloxy ester for several weeks. Atthe times indicated DNA was extracted and the average telomere lengthswere determined by the TRF method.

FIG. 2A shows U87 gb cells were treated with PMPG diisopropyloxy ester(ID#142715) (lane 4), 2′ deoxy, 3′ C6 phenyl-amide, 5′thiophosphateguanosine (lane 3), 2′ deoxy, 3′ azido, 5′ thiophosphate gunosine (lane2) or untreated (lane 1).

FIG. 2B shows Caki 1 cells treated five weeks with either 10 μM or 20 μMPMPG diisopropyloxy ester (ID#142715) (lanes 3 and 4) as compared withuntreated or DMSO treated cells (lanes 1 and 2).

FIG. 2C shows the treatment of A549 cells for seven weeks using either10 μM or 20 μM PMPG diisopropyloxy ester (lanes 3 and 4) as comparedwith untreated or DMSO treated cells (lanes 1 and 2). Cells weremaintained in the presence of compound and were passaged when confluent(1-2× per week). Cells were allowed to adhere to the plate (18-24 hr)before fresh compound was added. Genomic DNA was isolated from cells and3-6 μg digested with Hinf I and Rsa I. Samples were separated on a 0.8%agarose gel (1×TAE buffer). The gel was transferred to a positivelycharged nylon membrane and probed using TAGGG Telomere Length Assay Kit(Roche, Cat #12 209 136 001).

Treating several cancer cells in vitro for an extended period (4-6weeks) with the prodrug (R)-(((Guanine-9-yl) propan-2-oxy) methyl)phosphonic acid diisopropyloxy ester results in telomere shortening inthose cells, demonstrating not only that (R)-(((Guanine-9-yl)propan-2-oxy) methyl) phosphonic acid diisopropyloxy ester isefficiently converted to the triphosphate but more importantly that theactive molecule is added to its natural substrate (chromosome ends) incells.

E. Long-Term Treatment Assays

Long-term treatment of U87 glioblastoma cells with PMPG diisopropyloxyester reduces cell proliferation.

U87 glioblastoma cells were treated with either 10 μM or 20 μM PMPGdiisopropyloxy ester for 47 days. At the higher concentration of drugthe cells ceased to divide and the experiment was terminated. Growthcurves are shown (FIG. 3A). In FIG. 3B, equal cell numbers (75,000/well)were seeded following 5 weeks treatment with drug. Photos were taken 6days later. A reduction in cell proliferation has also been observed incancer cells in vitro suggesting that this reduction in growth is aconsequence of telomere shortening.

F. Inhibition of Human Tumor Growth in Animal Models

The abilities of the compounds to inhibit growth of human tumors inanimals will be tested. Athymic (nu/nu) mice will be inoculated withDU-145 tumor cells in both flanks. When the tumors (two tumors/mouse)reach 50-100 mm³ in size, the mice will receive oral administration ofPMPG diisopropyloxy ester on a daily basis. Mice will be sacrificedafter at least one week and the size of the tumors will be evaluated. Itis expected that mice treated with PMPG diisopropyloxy ester will havesmaller tumors than control mice.

Example 3. Screening of Compounds in Biochemical and Cell Based Assays

Screening of multiple compounds was accomplished by: (1) evaluating eachcompound as a dNTP and determining its ability to serve as a telomerasesubstrate and chain terminator by using a primer extension assay fortelomerase activity; (2) if a compound was a chain terminator, theprodrug version of this compound was examined using the ex vivo TRAPassay to determine if it scored as an inhibitor of telomerase byassessing: (a) whether the prodrug could be converted to a triphosphate;(b) the cellular IC50 using endogenous dGTP pool; and (c) cellularuptake. Additionally, long term cellular assays were used to assess acompound's ability to shorten telomeres and effect cell proliferation,with: (a) delayed effect on proliferation and (b) lack of cytotoxicityin (telomerase negative) primary cells or alt-transformed cells used aspositive indicators.

Materials and Methods

A. Primer Extension Assay for Telomerase Activity

Purified human telomerase was incubated with d(TTAGGG)₃ (SEQ ID NO: 1)and deoxy-nucleoside triphosphates for 90 minutes at 37° C. Allreactions were done in the presence of 200 dTTP and 10 μM dATP (50,000cpm/pmol a-³³P-dATP). Activity was determined by incubating affinitypurified telomerase extract with 1 33M primer [d(TTAGGG)₃] (SEQ ID NO:1), 200 33M dTTP, 50 33M dGTP, 10 33M dATP, 10 33Ci [α-³³P]dATP(2000-4000 Ci/mmol) in a buffer containing 50 mM (N-2-hydroxyethylpiperazine-N′-3 propanesulfonic acid)-NaOH pH 8.5, 1 mM MgCl₂, 1 mM DTT,5% glycerol, 0.5 mM EGTA and 100 mM KOAc in a final reaction volume of40 μl. Primer extension products were analyzed on a 15% polyacrylamidegel containing 7 M urea. A characteristic telomerase ladder has multiplebands. If a compound is not a substrate then (TTAGGG)₃TTA (21 nt) (SEQID NO: 2). If a compound is a chain terminator then (TTAGGG)₃TTAG* (22nt) (SEQ ID NO: 3).

B. Flash Plate Assay

Compound was pre-incubated with affinity purified telomerase extract,100 nM primer [5′ biotin-d(AATCCGTCGAGCAGAGTT)] (SEQ ID NO: 4), 120 μMdTTP, 20 μM dGTP, 10 μM dATP, in a buffer containing 50 mM Tris-Acetate(pH 8.2), 1 mM MgCl₂, 1 mM DTT, 0.5 mM EGTA and 150 mM KOAc, 5.4 nM³³P-labeled [d(CCCTAACCCTAACCCTAACCC)] (SEQ ID NO: 5 (6×10⁹ cpm/nmol) ina final reaction volume of 30 μl. Incubations were done for 90 minutesat 37° C. Following stoppage of the reaction with EDTA (10 mM), thereaction products were captured in a streptavidin coated 96 wellFlashPlate. After a 2 hour capture period for the primer, the plateswere washed five times (2×SSC, 0.1% SDS, 10 mM EDTA) and counted.

C. Telomere Repeat Fragment (TRF) Assay

Approximately 1-2 μg of genomic DNA from each cell line or tissue wasdigested by Hinf/RsaI for 2 hours at 37° C. Following DNA digestion, thegenomic DNA fragments were separated on a 0.8% agarose gel andtransferred to a nylon membrane by blotting. All membrane hybridizationand detection reagents were provided in the TeloTAGGG Telomere LengthAssay kit (Roche Diagnostics, Mannheim Germany). The blotted membraneswere hybridized with digoxigenin (DIG)-labeled PNA probe specific fortelomeric repeats, followed by incubation withanti-DIG-antibody-alkaline phosphatase according to the manufacturer'sinstructions. Alkaline phosphatase was quantified using thechemiluminescent substrate. Telomere lengths were calculated accordingto the manufacturer's instruction.

E. Ex-Vivo TRAP Assay

HT3 cells were treated for three days with increasing concentrations ofone or more of the indicated compounds ranging from 0.2 μM to 20 μM.Cells (HT3 RPMI+10% FBS) were incubated with compound for 24 hours,after which PSTS primer (phosphorothioate-d AATCCGTCGAGCAGAGTT (SEQ IDNO: 6) containing a 5′ Cy-5 label) (7.5 μM) and fresh compound wereadded for an additional 24 hours. Cells were lysed and endogenoustelomerase inactivated by heating (15 minutes, 75° C.). Products wereamplified by 30 cycles of PCR under TRAP conditions set forth in Kim etal., Science 266:2011, 1997 (which reference is incorporated herein inits entirety) and quantified.

F. Long-Term Treatment Assays

Indicated cell lines were treated with either 10 μM or 20 μM PMPGprodrug [(R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic aciddiisopropyloxy ester] (ID#142715) for 47 days. The cells were splitevery 5-7 days. At the higher concentration of drug the cells ceased todivide and the experiment was terminated.

Results

FIG. 4A is a photograph of a resulting gel from a primer extension assayrun in competition mode. From left to right, Lanes 1 and 2 (marked “−”)show a characteristic 6 nucleotide ladder. Lanes 3-8 contain increasingconcentrations of (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diphosphate (R-PMPGpp) (ID#142692), from 0.25 μM to 50 μM. Themolecule competes with dGTP and when measured in a cell free assay,using purified telomerase and dGTP concentrations of 50 μM, shows anIC50 of about 1.0 μM.

FIG. 4B shows (R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic aciddiphosphate (R-PMPGpp) (ID#142692) activity. The percent activity wasdetermined based on values obtained by PhosphoImager analysis of theintensity of the gel bands in FIG. 4A.

Together, FIGS. 4A and 4B show that R-PMPGpp (ID#142692) is efficientlyrecognized by human telomerase and added to the 3′ end of a telomericprimer, resulting in chain termination.

FIG. 5 shows an Ex Vivo TRAP assay with PMPG prodrug (ID#142715). Twoindependent assay results are shown for the same compound, shows theresults of an Ex Vivo TRAP assay. Telomerase inhibition is observed incells following the addition of PMPG prodrug [R-(((Guanine-9-yl)propan-2-oxy) methyl) phosphonic acid diisopropyloxy ester] (ID#142715).This compound is efficiently taken up by cells, is converted to theactive drug inside the cells, and competes with the endogenous dGTPpool.

FIG. 6 shows the growth curves for Caki-1 cells treated with 10 μM or 20μM PMPG prodrug [R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diisopropyloxy ester](ID#142715). The reduced growth of Caki-1cells manifests after a similar duration using PMPG prodrug (ID#142715).

The PMPG free acid (ID#142693) was acutely cytotoxic at a concentrationof 200 uM, but displayed minimal cytotoxicity at therapeutic doses (10μM and 20 μM).

FIG. 7 shows the growth curves for A549 cells treated with 10 μM or 20μM PMPG prodrug [R-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonicacid diisopropyloxy ester](ID#1427150) compared to 5 μM or 10 μMImetelstat (163 L). Growth inhibition is observed in the A549 cells,albeit with a significant lag. These results suggest cell growth wasaffected through telomere-related attrition rather than cytotoxicity.

FIG. 8A is a photograph of a resulting gel from a primer extensionassay. From left to right, Lane 1 (marked “−”) shows a characteristic 6nucleotide ladder. Lanes 2-5 contain increasing concentrations of(R)-(((Guanine-9-yl) propan-2-oxy) methyl) phosphonic acid diphosphate(R-PMPGpp) (ID#142692), from 0.4 μM to 50 μM. Lanes 6-9 contain from 0.4μM to 50 μM of PMIBeGpp (ID#142810).

FIG. 8B shows the dose response telomerase inhibition for R-PMPGpp(ID#142692) as compared to PMIBeGpp (ID#142810). The percent activitywas determined based on values obtained by PhosphoImager analysis of theintensity of the gel bands in FIG. 8A. PMIBeG is approximately two timesless potent than R-PMPGpp.

The PMIBeG free acid[(((2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methyl)allyl)oxy)methyl)phosphonicacid] (ID#142811) is less cytotoxic than PMPG, and showed no effect ontissue culture at concentrations of 200 μM when tested in RPE-64-tvcells.

FIG. 9 shows the growth curves for Caki-1 cells treated with 10 or 20 μMPMIBeG diisopropyloxy ester (ID#142820) or PMPG free acid (ID#142693).PMIBeG diisopropyloxy ester reduces cancer cell growth similar to PMPGfree acid.

FIG. 10 shows the gel resulting from a telomere repeat fragment assayusing A549 cells. Lane 1 is a ladder from 21.2 kb to 1.9 kb. Lanes 2 and3 have 10 μM and 5 μM, respectively, of PMPG free acid (ID#142693).Lanes 4 to 6 have 10 μM, 5 μM, and 2.5 μM, respectively, of PMPG prodrug(ID#142715). Lane 7 has 2 μM of Imetelstat (163 L). Lane 8 has 2 μM of amismatch. Lanes 9 and 10 have 20 μM and 10 μM, respectively, of PMIBeGfree acid[(((2-((2-amino-6-oxo-1H-purin-9(6H)-yl)methyl)allyl)oxy)methyl)phosphonicacid] (ID#142811). Indicated amounts each compound were added to A549cells. After approximately 70 population doublings, the cells wereharvested and telomere lengths determined using TRF. The estimatedaverage telomere lengths were: (1) MM/mismatch control: 7 kb; (2)Imetelstat/163 L: 4.3 kb; (3) PMPG prodrug (10 μM and 5 μM): 4 kb; (4)PMPG free acid (5 μM and 10 μM): 4 kb; (5) PMIBeG prodrug (50 μM): 4.3kb; (6) PMIBeG free acid (20 μM): 5 kb.

Telomere repeat fragment assays using immortal U87 cells were performed.10 μM or 20 μM PMIBeG prodrug (ID#142820) or PMPG prodrug (ID#142715)were added to the cells. Imetelstat (163 L) was used as a reference anda mismatch was used as a negative control. After approximately 20population doublings, the cells were harvested and telomere lengthsdetermined using TRF. The estimated average lengths were: (1) mismatchcontrol: 5 kb; (2) Imetelstat (163 L): 3.5 kb; (3) PMIBeG prodrug(ID#142820) at 20 μM: 4 kb; (4) PMIBeG prodrug (ID#142820) at 10 μM: 4.1kb; (5) PMPG prodrug (ID#142715): 3.5 kb.

Growth curves were prepared using immortal U87 cells treated with: 10 μMor 20 μM PMIBeG prodrug (ID#142820), 10 μM or 20 μM PMPG prodrug(ID#142715), or 2 μM Imetelstat (163 L). U87 cells for TRF assays wereharvested after approximately 20 cumulative population doublings. ThePMPG prodrug resulted in reduced growth of immortal U87 cells at boththe 20 μM and 10 μM concentrations.

Example 4: Inhibition of Human Tumor Growth in Animal Models

Materials and Methods

Low-passage Caki-1 cells were injected subcutaneously into the flank ofSCID hairless outbred (SHO) mice receiving 30 mg/kg of one of thefollowing via intraperitoneal injection: vehicle (PBS with 0.4% Tween20), Imetelstat (163 L), PMPG free acid (ID#142693), or PMPG prodrug(ID#142715). Calipers were used to measure tumor length and widthbi-weekly, and tumor volume was calculated. Once a tumor had grown >50mm³, the animal was randomly entered into a study group, either controlor treatment. Animals received 30 mg/kg compound via intraperitonealinjection for the duration of the study. The control vehicle was PBSwith 0.4% Tween 20. Tumor volume was measured bi-weekly, and animalswere sacrificed either when tumor size was >2000 mm³ or at defined studycompletion date.

At the conclusion of the study, a linear transformation of the tumorgrowth curves was performed. The slope of the growth curves for eachtreatment group was derived by using a linear mixed effects model. TheY-intercept was used to confirm random tumor starting size at the timeof animals entering into the study.

Results

FIG. 11 shows the plot of fitted growth curves (Imetelstat Day<41)obtained when low-passage Caki-1 cells were injected subcutaneously intothe flank of SCID hairless outbred (SHO) mice receiving 30 mg/kg ofvehicle (PBS with 0.4% Tween 20). Imetelstat. PMPG free acid (ID#142693)or PMPG prodrug (ID#142715) via intraperitoneal injection. The PMPG freeacid (ID#142693) and Imetelstat (163 L) used as a reference bothdisplayed a reduction in tumor growth compared to a vehicle control. Thelevel of reduction seen is consistent with the effects of sorafenib andsunitinib, both currently used kidney cancer targeted therapeutics (seeMiyake et al., Oncology Letters, Vol. 3: 1195-1202 (2012), which isincorporated in its entirety). PMPG prodrug (ID#142715) and vehicle wereindistinguishable in tumor growth.

The tumor volume data (in mm³) are in Table 5 below.

TABLE 5 Day Treatment 10 20 30 40 50 60 Vehicle 181.3 398.7 743.7 1245.81934.4 2839.2 Imetelstat 146.0 293.5 517.0 832.2 1254.8 1800.7 PMPG FreeAcid 165.1 330.8 581.3 934.3 1407.2 2017.6 PMPG Prodrug 170.7 377.5706.6 1186.6 1845.9 2713.0

The tumor volume data, expressed as a percentage of the tumor volume forthe mice injected with the control vehicle, are listed in Table 6 below.

TABLE 6 Day Treatment 10 20 30 40 50 60 Vehicle 100%  100%  100%  100% 100%  100%  Imetelstat 81% 74% 70% 67% 65% 63% PMPG Free 91% 83% 78% 75%73% 71% Acid PMPG Prodrug 94% 95% 95% 95% 95% 96%

Table 7 below shows that the linear transformed growth curves forImetelstat (163 L) and PMPGFree (ID#142693), as shown in FIG. 11,displayed statistically significant slope changes. The baselineslope/intercept was 0.0000. The starting tumor variability wasinsignificant.

TABLE 7 Standard Coefficient Error P Vehicle Intercept 3.960 0.1310.0000 Slope 0.169 0.009 0.0000 Intercept Imetelstat −0.074 0.190 0.6969PMPGFree 0.096 0.185 0.6054 PMPGProDrug −0.093 0.187 0.6204 SlopeImetelstat −0.032 0.014 0.0232 PMPGFree −0.027 0.013 0.0395 PMPGProDrug−0.001 0.013 0.9219

Table 8 below lists the EC50 values for both the PMPG Free Acid(ID#142693) and PMPG Prodrug (ID#142715).

TABLE 8 Compound EC50 (μM) PMPG Free Acid 11.7 PMPG Free Acid 2.5 PMPGFree Acid 1.5 PMPG Prodrug 1.2 PMPG Prodrug 3.1 PMPG Prodrug 0.81 PMPGProdrug 1.1

Table 9 lists the name, EC50 (μM) value as determined by primerextension assay, identification number, chemical structure and notesapplicable to screened compounds.

TABLE 9 Primer Ext. Assay EC50 Compound Primer Ext. Name (μM) ID#Chemical Structure Assay Notes PMPG free acid, R form 0.5-1.2 142692

Chain terminator. 6-thio-G- phosphonate (6-S-PMPG), R form   13.5 142761

Chain terminator. Similar affinity as PMPG in single nucleotide additionassay. PMPG free acid, S form 142751

Weak chain terminator. 6-S-PMPG (S) 142752

Weak chain terminator. PMPG(lin)    1.5 142848

Chain terminator. About 2X less potent than PMPG. PM(3)BG   10   142916

PM(2)BG >60   142915

FPMPG    7.5 142856

Chain terminator. TFPMPG 142850

Weak chain terminator. PMIBeG, free acid    1.4 142810

Chain Terminator. PMIBaG, R form 142860

Weak chain terminator. PMIBaG (mix R,S)    4.4 142846

Chain terminator. PMP(2)G    3.1 142879

Chain terminator. PMPG (dimethyl) 142891

Weak chain terminator. mercapo- PMPG, R form 142823

Weak chain terminator. PMIBaG, R form 142860

Weak chain terminator.

Table 10 lists the compound identification (ID) number, EC50 asdetermined by Ex Vivo TRAP assay, chemical structure, and notesapplicable to screened compounds.

ExVivo TRAP EC50 Com- (μM)- pound 3 Day Name ID# Assay ChemicalStructure PMPG free acid, R form 142693 11.7

PMPG prodrug, R form 142715  1.2,  3.1

6-thio-G- phosphonate (6-S- PMPG), R form 142694  7.54

PMPG free acid, S form 142753

PMPG prodrug, S form 142748

FPMPG 142855

TFPMPG 142834

PMIBeG free acid 142811  2.6

PMPeG, prodrug 142820  3.9, 17.8

Saturated PMIBeG phosphonate S enantiomer 142866

PMIBeG spermidine 142873  6.7

PMPG spermidine, R form 142812  5.0

PMIBaG, R form 142859

PMIBaG (mix R,S) 142844

mercapo- PMPG, R form 142821

1101C compound B. 142824

PMPG spermine, R form 142779

PMPG-C10 amidate 142962  1.44

bis-PMPG spermine 142942  1.84

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation. Allpublications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. In particular,all publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A compound of formula (III):

wherein R¹ and R² are independently selected from —NR^(1a)R^(1b) andOR^(1c); wherein R^(1a) and R^(1b) are independently selected fromhydrogen, optionally substituted C₁₋₂₀alkyl, optionally substitutedpolyamine, and CH(R^(1d))—C(O)OR^(1e), wherein R^(1d) is selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, and substitutedheteroarylalkyl; and R^(1e) is hydrogen or C₁₋₆alkyl; R^(1c) is selectedfrom hydrogen, alkyl, and aryl; wherein at least one of R¹ and R² is—NR^(1a)R^(1b); R^(3a) and R^(3b) are independently selected fromhydrogen and halo; W is O, S, or NH; R^(4a) and R^(4b) are independentlyselected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionallysubstituted C₁₋₂ alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃,or halogen; R^(5a) and R^(5b) are independently selected from hydrogen,—OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, whereinalkyl is substituted with —OH, —NH₂, N₃, or halogen; R^(6a) and R^(6b)are independently selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, andoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen; R⁷ is hydrogen or fluoro; and X is O, S, orNH; and salts, hydrates, solvates, and tautomers, thereof.
 2. Thecompound of claim 1, wherein: R¹ is different from R²; or one of R¹ andR² carries a positive charge and the other carries a negative charge; orR¹ is —NR^(1a)R^(1b) and R² is OR^(1c).
 3. The compound of claim 1,wherein: one of R^(1a) and R^(1b) is C₁₋₂₀alkyl and R² is OH; or one ofR^(1a) and R^(1b) is a polyamine and R² is OH; or one of R^(1a) andR^(1b) is —(CH₂)_(n)NH(CH₂)_(n)NHR^(x) wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂ and n is independently a number from 2 to 4; or one ofR^(1a) and R^(1b) is —(CH₂)_(n)NHR^(x) wherein R^(x) is hydrogen or—(CH₂)_(n)NH₂ and n is independently a number from 2 to 4; or one ofR^(1a) and R^(1b) is —CH(R^(1d))—C(O)OR^(1e) and R² is OH.
 4. Thecompound of claim 1, wherein: R^(1d) is selected from hydrogen, alkyl,substituted alkyl, heteroaryl, and substituted heteroaryl; or R^(1d) isa positively charged amino acid side chain.
 5. The compound of claim 1,wherein: R^(3a) and R^(3b) are hydrogen; or one of R^(3a) and R^(3b) ishalo.
 6. The compound of claim 1, wherein W is O.
 7. The compound ofclaim 1, wherein R^(4a) and R^(4b) are hydrogen.
 8. The compound ofclaim 1, wherein: one of R^(5a) and R^(5b) is —OH; or one of R^(5a) andR^(5b) is selected from —NH₂ and N₃; or one of R^(5a) and R^(5b) is—CH═CH₂; or one of R^(5a) and R^(5b) is C₁₋₂ alkyl; or one of R^(5a) andR^(5b) is C₁₋₂ alkyl, substituted with —OH, —NH₂, or N₃.
 9. The compoundof claim 1, wherein R^(6a) and R^(6b) are hydrogen.
 10. The compound ofclaim 1, wherein R⁷ is hydrogen.
 11. The compound of claim 1, wherein Xis O.
 12. A pharmaceutical composition comprising the compound of claim1 and a pharmaceutically acceptable excipient.
 13. A method of treatingbreast or pancreatic cancer in an individual by administering aneffective amount of the compound of claim
 1. 14. The method of claim 13,wherein the cancer is pancreatic cancer.
 15. The method of claim 13,wherein the cancer is breast cancer.
 16. A compound of formula (IV):

wherein X¹ is NH or O; X² is NH or O; R³⁰ is hydrogen, optionallysubstituted C₁₋₂₀alkyl, optionally substituted C₂₋₂₀alkenyl, oroptionally substituted C₂₋₂₀alkynyl; R¹ is selected from —NR^(1a)R^(1b)and OR^(1c); wherein R^(1a) and R^(1b) are independently selected fromhydrogen, optionally substituted C₁₋₂₀alkyl, optionally substitutedpolyamine, and —CH(R^(1d))—C(O)OR^(1e), wherein R^(1d) is selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, and substitutedheteroarylalkyl; and R^(1e) is hydrogen or C₁₋₆alkyl; R^(1c) is selectedfrom hydrogen, alkyl, and aryl; R^(3a) and R^(3b) are independentlyselected from hydrogen and halo; W is O, S, or NH; R^(4a) and R^(4b) areindependently selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, andoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen; R^(5a) and R^(5b) are independently selectedfrom hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen;R^(6a) and R^(6b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen; R⁷ is hydrogen or fluoro;and X³ is O, S, or NH; and salts, hydrates, solvates, and tautomers,thereof.
 17. The compound of claim 16, wherein X¹ and X² are O.
 18. Thecompound of claim 16, wherein R³⁰ is C₁₋₂₀alkyl.
 19. The compound ofclaim 16, wherein: R¹ carries a positive charge or a negative charge; orR¹ is —NR^(1a)R^(1b).
 20. The compound of claim 19, wherein: one ofR^(1a) and R^(1b) is C₁₋₂₀alkyl; and R² is OH; or one of R^(1a) andR^(1b) is a polyamine and R² is OH; or one of R^(1a) and R^(1b) is—(CH₂)_(n)NH(CH₂)_(n)NHR^(x), wherein R^(x) is hydrogen or —(CH₂)—NH₂and n is independently a number from 2 to 4; or one of R^(1a) and R^(1b)is —(CH₂)_(n)NHR^(x), wherein R^(x) is hydrogen or —(CH₂)_(n)NH₂ and nis independently a number from 2 to 4; or one of R^(1a) and R^(1b) is—CH(R^(1d))—C(O)OR^(1e), and R² is OH.
 21. The compound of claim 16,wherein; R^(1d) is selected from hydrogen, alkyl, substituted alkyl,heteroaryl, and substituted heteroaryl; or R^(1d) is a positivelycharged amino acid side chain.
 22. The compound of claim 16, wherein:R^(3a) and R^(3b) are hydrogen; or one of R^(3a) and R^(3b) is halo. 23.The compound of claim 16, wherein W is O.
 24. The compound of claim 16,wherein R^(4a) and R^(4b) are hydrogen.
 25. The compound of claim 16,wherein: one of R^(5a) and R^(5b) is —OH; or one of R^(5a) and R^(5b) isselected from —NH₂ and N₃; or one of R^(5a) and R^(5b) is —CH═CH₂; orone of R^(5a) and R^(5b) is C₁₋₂ alkyl; or one of R^(5a) and R^(5b) isC₁₋₂ alkyl, substituted with —OH, —NH₂, or N₃.
 26. The compound of claim16, wherein R^(6a) and R^(6b) are hydrogen.
 27. The compound of claim16, wherein R⁷ is hydrogen.
 28. A pharmaceutical composition comprisingthe compound of claim
 16. 29. A method of treating breast or pancreaticcancer in an individual by administering an effective amount of thecompound of claim
 16. 30. The method of claim 29, wherein the cancer ispancreatic cancer.
 31. The method of claim 29, wherein the cancer isbreast cancer.
 32. A method of treating breast or pancreatic cancer inan individual by administering an effective amount of a compound offormula (VI):

wherein R¹ and R² are independently —OCH(R^(1b))OC(O)OR^(1a); whereinR^(1a) and R^(1b) are independently selected from hydrogen andC₁₋₅alkyl; R^(3a) and R^(3b) are independently selected from hydrogenand halo; W is O, S, or NH; R^(4a) and R^(4b) are independently selectedfrom hydrogen, —OH, —NH₂, N₃, —CH═CH₂, and optionally substituted C₁₋₂alkyl, wherein alkyl is substituted with —OH, —NH₂, N₃, or halogen;R^(5a) and R^(5b) are independently selected from hydrogen, —OH, —NH₂,N₃, —CH═CH₂, and optionally substituted C₁₋₂ alkyl, wherein alkyl issubstituted with —OH, —NH₂, N₃, or halogen; R^(6a) and R^(6b) areindependently selected from hydrogen, —OH, —NH₂, N₃, —CH═CH₂, andoptionally substituted C₁₋₂ alkyl, wherein alkyl is substituted with—OH, —NH₂, N₃, or halogen; R⁷ is hydrogen or fluoro; and X is O, S, orNH; and salts, hydrates, solvates, and tautomers, thereof.
 33. Themethod of claim 32, wherein the cancer is pancreatic cancer.
 34. Themethod of claim 32, wherein the cancer is breast cancer.